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BIOLOGICAL  STUDIES 


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BIOLOGICAL    STUDIES 


BY  THE  PUPILS  OF 


WILLIAM  THOMPSON  SEDGWICK 


Published  in   Commemoration 

OF  THE  Twenty-fifth  Anniversary 

OF  HIS  Doctorate 


BOSTON 

JUNE,     1906 


PRINTED    AT 

THE    UNIVERSITY  OF  CHICAGO   PKESS 

1906 


THIS     BOOK    IS     DEDICATED     BY     HIS     PUPILS     TO 

WILLIAM  THOMPSON  SEDGWICK 

TO  EXPRESS  THEIR  REGARD  AND  ADMIRATION  FOR 
HIM  AS  A  FRIEND,  TEACHER,  INVESTIGATOR,  AND 
PUBLIC-SPIRITED  CITIZEN,  AND  ALSO  TO  AFFIRM 
THEIR  LOYALTY  TO  THE  IDEALS  FOR  WHICH  HE 
HAS    ALWAYS    STOOD. 


'T 


A  C- 


TABLE   OF   CONTENTS. 

PAGE 

Calkins,  Gary  N.     Paramecium  aurelia  and  Paramecium  caudatum    -  i-io 

Dyar,   Harrison    G.     The   Life-History   of   a   Cochlidian   Moth — 

Adoneta  bicaudata  Dyar 11-19 

Fuller,  George  W.     Experimental  Methods  as  Applied  to  Water- 

and  Sewage-Works  for  Large  Communities        .        -        -        -        20-35  , 

Leighton,  Marshall  O.     The  Futility  of  a  Sanitary  Water  Analysis 

as  a  Test  of  Potability 36-53 

Whipple,  George  C.    The  Value  of  Pure  Water     -        .        -        -        54-80 

Mathews,  A.  P.     A  Contribution  to  the  General  Principles  of  the 

Pharmacodynamics  of  Salts  and  Drugs 81-118 

Stiles,  Percy  G.,  and  Milliken,  Carl   S.     An  Instance  of  the 

Apparent  Antitoxic  Action  of  Salts 119-123 

Jordan,  Edwin  O.     Experiments  with  Bacterial  Enzymes  -        -        -     124-145 

.^WiNSLOW,  C.-E.  A.,  and  Rogers,  Anne  F.    A  Statistical  Study  of 

Generic  Characters  in  the  Coccacea; 146-207 

^Prescott,  Samuel  C.  The  Occurrence  of  Organisms  of  Sanitary  Sig- 
nificance on  Grains       -.- 208-222 

^^Gage,  Stephen  DeM.  A  Study  of  the  Numbers  of  Bacteria  Develop- 
ing at  Different  Temperatures  and  of  the  Ratios  between  Such 
Numbers  with  Reference  to  Their  Significance  in  the  Interpreta- 
tion of  Water  Analysis        ....----     223-257 

'  WiNSLOW,  C.-E.  A.,  AND  Lochridge,  E.  E.  The  Toxic  Effect  of  Cer- 
tain Acids  upon  Typhoid  and  Colon  Bacilli  in  Relation  to  the 
Degree  of  Their  Dissociation       -.--..-     258-282 

Phelps,  Earle  B.     The  Inhibiting  Effect  of  Certain   Organic  Sub- 
stances upon  the  Germicidal  Action  of  Copper  Sulphate     -        -     283-291 

Jackson,  Daniel  D.     A  New  Solution  for  the  Presumptive  Test  for 

Bacillus  coli         .         .        - --     292-299 

Ayers,  S.  Henry.     B.  coli  in  Market  Oysters 300-303 

Wadsworth,  Augustus.     Studies  on  Simple  and  Differential  Methods 

of  Staining  Encapsulated  Pneumococci  in  Smear  and  Section  -        -    304-312 

Kendall,  Arthur  I.    An  Apparatus  for  Testing  the  Value  of  Fumi- 
gating Agents 3i3~320 

vii 


viii  Table  of  Contents 

PAGE 

Hough,  Theodore,  and  Ham,  Clara  E.  The  Effect  of  Subcutaneous 
Injections  of  Water,  Ringer's  Fluid,  and  Ten  Per  Cent  Solution 
of  Ethyl  Alcohol  upon  the  Course  of  Fatigue  in  the  Excised  Mus- 
cles of  the  Frog   -        -        .    321-326 

RiCKARDS,  Burt  R.  Notes  on  a  Case  of  Apparent  Pulmonary  Tu- 
berculosis Associated  with  the  Constant  Presence  of  Diphtheria- 
Like  Organisms  in  the  Sputum     -------    327-329 


PARAMECIUM  AURELIA   AND  PARAMECIUM  CAU- 
DA TUM* 

Gary  N.  Calkins,  Ph.D. 

At  the  present  time,  when  the  subject  of  mutations  and  species 
is  discussed  on  every  hand,  and  when  every  eye  is  keenly  on  the 
alert  for  new  evidence  among  animals  and  plants,  a  sudden  trans- 
formation of  one  known  species  into  another  known  species  is  of 
interest.     Such  an  incident  has  recently  come  under  my  observa- 
tion; a  Paramecium  caudatum  became  P.   aurelia,   and   remained 
so   for   about  45    generations,   when  it    reverted   to  P.   caudatum. 
Apart  from  the  facts  of  the  change,  which  in  itself  is  of  obvious 
importance  from   the  standpoint  of  cellular   biology,  the  essential 
question  to  consider  is  whether  these  two   species  are  sufficiently  well 
defined  to  justify  their  separation.     If  not,  then  the  experiments  and 
the  changes  indicated  have  less  bearing  on  the  general  problem  of  mu- 
tation than  upon  the  problems  of  cell  physiology.     If  they  are  suffi- 
ciently distinct,  then  we  have  in  this  incident  an  interesting  case  of  mu- 
tation.   I  personally  believe  that  the  slight  differences  that  distinguish 
the  two  types  of  Paramecium  are  not  of  specific  value,  and  hold^that 
P.  caudatum  should  be  regarded  as  a  mere  variant  of  P.  aurelia. 
Paramecium  aurelia  was  the  name  given  by  Muller,  in  his  general 
work  on  the  Protozoa  in  1773,  to  the  ciliated  organism  which  had 
been  known  as  the  "slipper  animalcule."     Several  different  species 
of  Paramecium  were  created  by  Ehrenberg  in  1838,  and  described 
in  his  work  on  the  Injusionsthierchen.     Most  of  these  have  been 
sifted  out  into  other  genera,  and  only  three  have  remained,  P.  bur- 
saria,   P.   aurelia,   and  P.   caudatum.     Paramecium  caudatum  and 
P.  aurelia  have  been  united  into  a  single  species  by  the  majority 
of  observers  subsequent  to  Ehrenberg,  on  the  ground  that  the  differ- 
ences upon  which  Ehrenberg  had  based  his  species  were  inadequate. 
The  number  of  species  was  thus  reduced  to  two,  and  the  names 
used  were  P.  aurelia  and  P.  bursaria,  the  former  having  been  given 
originally  by  Muller.     Maupas,  however,  in  1887-89,  and  R.  Hert- 

*  Received  for  publicatioQ  March  17,  igo6. 


jqtOrERIT  IMAM 

K.  C  State  C»ll*f 


2  Gary  N.  Calkins 

wig  in  1889,  discovered  a  difference  in  the  two  forms  which  appeared 
to  have  specific  value,  and  since  then  the  two  species  in  question, 
caudaiiim  and  aurelia,  have  been  generally  accepted  as  "good" 
species. 

Paramecium  aurelia,  according  to  Maupas,  differs  from  P.  cau- 
datum  in  the  following  points:  It  is  smaller  (70  to  290  ft,  as  against 
120  to  325  /i  in  P.  caudatum);  its  posterior  end  is  rounded,  while 
P.  caudatum  has  an  attenuated  end  (hence  caudatum);  it  has  two 
small  micronuclei  (3  to  5  /a  in  diameter),  while  P.  caudatum  has 
but  one  (8  to  10 /tt);  in  conjugation  its  macronucleus  becomes  "rib- 
bon "-shaped  at  an  earlier  period  than  in  P.  caudatum;  and  after 
conjugation  its  cleavage  nucleus  gives  rise  to  four  corpuscles,  whereas 
in  P.  caudatum  there  are  eight.  In  deciding  which  of  these  forms  to 
call  caudatum  and  which  aurelia,  Maupas  could  not  determine  which 
type  Miiller  had  seen,  and  went  back  therefore  only  to  Ehrenberg, 
who'  in  naming  P.  caudatum  had  noted  the  attenuated  posterior 
end.  Hence  it  turns  out  that  the  more  common  form  of  Para- 
mecium has  become  widely  known  as  P.  caudatum,  while  the  less 
common  form  bears  the  original  name  P.  aurelia.  If  the  two  are 
only  variants  of  the  same  species,  it  follow^s  from  the  rules  of  zo- 
ological nomenclature  that  the  common  and  well-known  name  Para- 
mecium caudatum  must  be  given  up  and  P.  aurelia  substituted. 
That  this  must  be  the  case  follows,  as  I  believe,  from  the  observa- 
tions here  described.  In  the  following  description  the  names  P. 
caudatum  and  P.  aurelia  will  be  used  for  those  variants  of  the  organ- 
isms which  agree  with  Maupas'  specific  characteristics. 

On  March  11,  1905,  four  pairs  of  conjugating  Paramecium 
caudatum  were  isolated  from  a  culture  that  had  been  running  for 
some  weeks  in  the  laboratory.  Each  pair  was  confined  in  a  hollow 
ground  slide  in  a  medium  of  hay  infusion  made  the  previous  day 
by  boihng  a  small  quantity  of  hay  in  tap  water.  The  usual  period 
of  conjugation  is  from  18  to  24  hours,  and  by  the  following  day 
all  of  the  pairs  had  separated,  and  the  different  individuals  were 
swimming  about  freely  in  the  hay  infusion  as  apparently  normal 
ex-conjugants.  Each  individual  was  isolated  and  fed  on  hay  infu- 
sion, and  each  became  the  progenitor  of  a  more  or  less  extended  Hne 
of  Paramecia,  the  method  followed  being  the  same  as  that  described 


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4  Gary  N.  Calkins 

in  a  previous  publication.  As  in  previous  work,  the  number  of 
divisions  was  recorded  each  day  for  each  of  the  ex-conjugants.  In 
addition  to  the  ex-conjugants,  one  individual  from  the  original 
culture  which  had  not  conjugated,  was  isolated  at  the  same  time 
to  serve  as  a  sort  of  control.  This  line  was  designated  X;  the  others, 
A-B,  C-D,  E-F,  G-H,  etc. 

The  early  history  of  the  division  rate  for  all  of  these  forms  is 
given  in  the  accompanying  table. 

The  interest  in  the  present  paper  centers  in  the  first  pair,  A-B, 
for  it  was  in  this  line  of  cultures  that  the  aurelia  form  appeared. 
B  died  after  a  few  days,  but  A  recovered  from  the  shock  of  reor- 
ganization and  soon  began  to  divide  at  a  slow  rate.  As  is  the  cus- 
tom in  such  work,  one  of  the  daughter-cells  from  an  early  division 
was  killed  and  stained,  to  determine  if  conjugation  had  been  nor- 
mally completed.  The  preparation  shows  that,  instead  of  reor- 
ganizing with  one  micronucleus  characteristic  of  P.  caudatum,  this 
individual  had  two,  but  otherwise  appeared  normal.  From  time 
to  time  after  this  individuals  from  this  line  of  cultures  were  fixed 
and  stained,  and  these  preparations  give  a  good  history  of  the 
nuclear  conditions  at  different  periods.  Some  of  these  specimens 
are  shown  in  photographs  on  Plate  i.  Fig.  i  represents  an  individ- 
ual in  the  third  generation  after  conjugation,  with  four  micronuclei 
(already  divided  for  the  ensuing  generation)  and  the  as  yet  incom- 
plete macronucleus.  Fig.  2  represents  an  individual  in  the  24th 
generation;  Fig.  3,  one  in  the  43d;  Fig.  4,  one  in  the  46th;  Fig.  6 
shows  two  micronuclei  in  the  end  stage  of  division,  the  daughter- 
halves  are  connected  by  a  delicate  thread  not  seen  in  the  photograph ; 
Fig.  7,  finally,  represents  an  individual  in  the  220th  generation  after 
complete  recovery  of  the  P.  caudatum  condition.  The  change  from 
the  one  form  to  the  other  occurred  during  the  first  two  weeks  in  May. 
Figs.  3,  4,  and  5  represent  three  individuals  from  the  same  ancestor 
in  the  43d  generation.  The  first  two  individuals  have  each  two 
micronuclei,  the  third  has  only  one.  All  of  these  micronuclei  show 
a  marked  increase  in  size  over  those  shown  in  Fig.  2  for  example, 
representing  an  individual  in  the  24th  generation. 

During  the  month  of  May  and  until  three  months  after  the  cul- 
ture was  started,  individuals  appeared  here  and  there  with  but  one 


Paramecium  Aurelia  and  Paramecium  Caudatum 


5 


micronucleus,  while  the  majority  of  them  killed  at  this  time  appeared 
with  one  of  the  micronuclei  larger  than  the  other.  By  the  end  of 
June  none  of  the  P.  aurelia  forms  were  to  be  found,  and  this  culture, 
like  the  other  cultures  started  at  the  same  time  (G  and  X),  contained 
forms  with  only  one  micronucleus;  Paramecium  aurelia  had  become 
P.  caudatum  again.  This  occurred  between  the  45th  and  the  70th 
generations,  and  the  effect  of  this  change  upon  the  vigor  of  the  race 
is  evident  from  the  remarkable  rise  which  the  accompanying  curve 
representing  the  relative  division  rate  takes  at  this  period  (see  curve). 


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Curves  showing  one  nearly  complete  cycle,  and  the  variations  in  -vitality  as  meas- 
ured by  the  division  rate  for  ex-conjugants  of  the  A  series  (solid  line)  and  the  G  series 
(broken  line).  The  ordinates  represent  the  average  number  of  divisions  in  lo-day 
periods.  The  rise  in  the  A  curve,  as  indicated  in  the  first  lo  days  in  June,  marks 
the   recovery  of  the  uninucleate   condition  characteristic  of  Paramecium   caudatum. 

Maupas  held  that  the  two  species  of  Paramecium  can  be  readily 
distinguished  by  the  characteristics  given  above.  If  we  examine 
these  characters  in  the  light  of  the  experience  with  cultures,  we  find 
that  they  cannot  hold  good.     For  example,  the  relative  size  of  the 


6  Gary  N.  Calkins 

two  forms  which  Maupas,  and  with  him  more  recently  Simpson, 
held  to  be  a  distinctive  feature,  loses  all  value  when  considered 
critically,  while  size  relations  in  general  are  absolutely  untrust- 
worthy in  settling  questions  of  species.  The  variations  which  a 
species  of  Paramecium  passes  through  under  different  conditions 
of  vitality  are  so  great,  and  last  for  such  long  periods,  that  no  infer- 
ence can  be  drawn  from  cell  dimensions  at  any  given  time.  Size 
depends,  apparently,  upon  two  factors — the  relative  vitality,  and 
the  rate  of  cell  division — and  these  two  may  probably  be  merged 
into  one,  which  may  be  called  the  potential  of  vitality.  Parame- 
cium caudatum  under  different  conditions  shows  wide  variations 
in  size.  When  taken  directly  from  the  natural  habitat,  where  food 
is  not  overabundant,  they  are  large,  measuring  on  the  average 
315/14.  The  same  forms  cultivated  on  hay  infusion,  with  its  rich 
food  content,  multiply  rapidly,  and  do  not  grow  individually  to 
the  same  size  as  the  "wild"  form.  These  measure  on  the  average 
(18  individuals  killed  at  different  periods  of  the  cycle)  only  206  /*, 
with  variations  from  180  to  224  /x  when  the  division  rate  was  rela- 
tively high — two  divisions  per  day.  When  the  potential  of  vitality 
is  nearly  exhausted  and  the  division  rate  is  low,  a  similar  small 
size  is  noticed;  but  at  such  a  time  it  is  obviously  due  to  a  different 
cause,  probably  a  loss  of  metabolic  energy.  The  same  differences 
are  noticed  in  P.  aurelia  at  different  periods  of  vitality,  and  the 
impossibility  of  considering  size  relations  as  of  specific  value  is 
clearly  estabhshed.  During  the  first  45  generations  of  P.  aurelia 
the  division  rate  averaged  only  eight-tenths  of  a  division  per  day, 
which  is  lower  than  that  for  the  G  and  X  lines,  which  averaged 
one  and  one-tenth  divisions.  Eighteen  individuals  killed  at  differ- 
ent periods  of  the  culture  were  measured  during  these  45 
generations,  and  the  average  length  was  224  /a,  with  variations 
from  168  to  256  /A.  After  the  loss  of  one  of  the  micronuclei  the 
♦>  division  rate  increased  to  the  remarkable  rate  of  2 . 2  per  day  on 
the  average  for  a  period  of  four  months,  when  vitality  waned. 
During  this  period  of  rapid  multiplication  the  size  averaged  only 
178  ft  with  variations  from  148  to  212  /i.  At  this  period  the  organ- 
isms in  culture  would  have  been  identified  by  any  microscopist  as 
P.   caudatum,   although  more  than  40  /i,   on  the  average,  smaller 


Paramecium  Aurelia  and  Paramecium  Caudatum  7 

than  the  one  which  would  be  classed  as  P,  aurelia,  while  the  latter, 
in  turn,  measured  90  /*  less,  on  the  average,  than  wild  P.  caudatum. 
It  is  quite  apparent,  therefore,  that  size  cannot  be  taken  as  a  diag- 
nostic character  in  the  present  case;  and  this  is,  after  all,  only  the 
application  of  a  well-known  principle  in  zoological  taxonomy. 

The  pointed  condition  of  the  posterior  end,  also,  in  P.  caudatum 
is  likewise  transitory,  and  may  or  may  not  be  present  in  forms  which 
agree  in  all  other  characters.  Pointed  Paramecium,  if  isolated, 
and  the  descendants  watched  for  four  or  five  generations,  as  I  have 
done,  will  lose  this  characteristic  and  will  become  rounded  and 
blunt  at  the  posterior  end  (cf.  Figs.  3,  4,  and  7). 

The  sluggishness  which  Simpson  advanced  as  a  specific  char- 
acter of  P.  aurelia  is  purely  a  physiological  condition,  depending 
upon  the  vitality  at  a  given  time,  and  is  as  much  characteristic  of 
P.  caudatum  as  of  P.  aurelia. 

The  breaking-up  of  the  macronucleus  at  an  earlier  period  in 
conjugation,  which  Maupas  considered  a  diagnostic  feature  of 
P.  aurelia,  may  also  be  due  to  physiological  conditions.  I  cannot 
write  definitely  on  this  point,  as  I  have  had  no  experience  with  con- 
jugating forms  of  P.  aurelia.  By  itself  it  would  not  constitute  a 
diagnostic  characteristic  of  sufficient  value  to  determine  a  species. 
So,  too,  the  other  characteristic  feature  of  conjugating  forms,  named 
by  Maupas,  the  number  of  corpuscles  into  which  the  fertilization 
micronucleus  divides,  would  be  dependent  upon  the  number  of 
micronuclei  present,  and  would  amount  to  the  same  thing  in  either 
case,  if  each  of  the  two  micronuclei  of  P.  aurelia  forms  four  micro- 
nuclei,  as  Maupas  describes.  The  experience  which  I  have  de- 
scribed above  of  the  presence  of  two  micronuclei  after  conjugation 
in  forms  which  had  only  one  before,  indicates  that  eight  corpuscles 
characteristic  of  P.  caudatum  were  also  formed  here,  but  were 
resolved  into  a  binucleate  instead  of  a  uninucleate  condition.  This 
characteristic,  therefore,  cannot  be  termed  specific. 

Apart  from  the  purely  physiological  characteristics  which  have 
little  or  no  value  in  classification,  there  remains  only  the  one  specific 
feature  to  justify  the  attempt  to  separate  P.  aurelia  and  P.  caudatum, 
viz.,  the  presence  of  two  and  one  micronuclei  respectively.  My 
experiments  show  that   this,   too,   is  inadequate,   for  P.  caudatum 


8  Gary  N.   Galkins 

may  become  P.  aurelia,  and  P.  aurelia  may  in  time  lapse  again 
into  P.  caudatiim.  It  is  to  be  inferred  from  this  that  the  various 
forms  which  have  been  described  as  P.  aurelia  are  in  reahty  only 
"sports"  of  P.  caudaium,  and  if  such  "sports"  are  unable  to  keep 
up  by  inheritance  the  characteristic  structural  differences  which 
distinguish  them  from  the  ancestral  form,  although  this  is  main- 
tained for  the  long  period  of  45  generations,  they  cannot  be 
considered  a  "good  species."  On  the  other  hand,  R.  Hertwig 
gives  some  evidence,  not  conclusive,  however,  to  show  that  P.  aure- 
lia, after  conjugation,  reorganizes  with  two  micronuclei. 

It  must  be  admitted  that  one  experience  of  this  kind  may  be 
insufficient  to  throw  out  a  species  that  has  appeared  to  be  so  well 
established.  It  may  be  that  my  observation  was  made  on  a  chance 
abnormality  which  paralleled  P.  aurelia,  and  that  the  real  P.  aurelia 
retains  its  integrity  as  a  species.  Personally,  however,  I  do  not 
believe  it,  and  am  reasonably  confident  that  such  abnormalities 
may  be  of  frequent  enough  occurrence  in  nature  to  account  for  the 
numerous  descriptions  of  P.  aurelia  that  have  been  given.  Forty 
generations  is  a  long  series  for  an  abnormality  to  be  transmitted, 
and  the  number  of  individuals  represented  by  2  to  the  40th 
power  (as  my  culture  represents),  allowing  for  natural  loss  through 
enemies,  etc.,  would  provide  enough  specimens  with  this  abnor- 
mality to  justify  the  belief  that  it  is  normal.  On  the  other  hand, 
it  cannot  be  stated  that  P.  aurelia  is  a  well-established  species.  It 
is  relatively  rare  in  nature;  its  specific  character  has  been  con- 
tested by  such  eminent  authorities  as  BiitschH,  Engelmann,  Bal- 
biani.  Stein,  Koelhker,  and  Gruber;  while  Maupas  and  Hertwig 
succeeded  in  establishing  it  as  a  species  only  on  the  slender  basis 
given  above.  My  one  experience  with  this  culture  is  strong  enough, 
as  I  believe,  to  reanimate  the  old  skepticism,  and  to  justify  us  in 
abandoning  either  P.  aurelia  or  P.  caudaium.  The  latter  is  the 
more  recent  name  given  by  Ehrenberg,  and  according  to  the  rules 
of  priority  must  be  replaced  by  Paramecium  aurelia,  the  name  applied 
by  O.  F.  Mijller  to  the  "slipper  animalcule." 

The  physiological  features  presented  by  this  experiment  give 
some  interesting  data  upon  the  vitality  and  nuclear  relations.  The 
curve  shows  that  a  sudden  rise  in  vigor  accompanied  the  return 


Paramecium  Aurelia  and  Paramecium  Caudatum  9 

to  the  normal  through  the  loss  of  one  of  the  two  nuclei.  Although 
it  is  difficult  to  estimate  accurately  the  relative  volumes  of  nucleus 
and  cell  body,  some  idea  of  the  relationship  can  be  given  by  meas- 
uring the  two  dimensions  of  the  surfaces  exposed.  The  surface 
of  the  macro-  or  micronucleus  obtained  by  multiplying  the  two 
dimensions  exposed,  have  a  measurable  relation  to  the  total  surface 
of  the  organism,  and  in  a  rough  way  this  relationship  represents 
the  relative  volumes.  Measured  in  this  way,  it  was  found  that 
in  resting,  vegetative  cells  of  P.  aurelia  the  relation  on  the  average 
of  both  micronuclei  together  to  the  whole  organism  is  as  1:717  (or 
of  the  single  micronucleus  to  total  body  as  1:1,434).  In  dividing 
cells  this  proportion  rises  to  the  ratio  of  i :  608  (or  for  the  single 
micronucleus  as  1:1,216).  In  the  P.  caudatum  stage,  after  the  loss 
of  one  micronucleus,  the  ratio  of  the  micronucleus  to  the  entire 
cell  is  expressed  by  the  ratio  of  1:648  for  resting  cells  and  1:440 
for  dividing  cells. 

The  macronucleus  is  generally  considered  to  be  the  primary 
agent  in  constructive  processes  of  the  cell,  and  therefore  of  the  first 
importance  in  considering  the  growth  and  division  energy.  In 
the  P.  aurelia  phase,  when  the  division  rate  was  low,  the  average 
relation  of  the  macronucleus  to  the  whole  body  was  as  i :  43,  with 
wide  variations  from  1:16  to  1:68.  In  the  P.  caudatum  phase, 
with  one  micronucleus,  the  proportion  of  the  macronucleus  to  the 
entire  cell  fell  to  1:50  on  the  average,  with  variations  from  i :  39 
to  1:82.  The  difference  is  not  great,  and  may  well  fall  within  the 
limits  of  experimental  error,  and  it  is  reasonable  to  infer  that  the 
physiological  differences  which  are  evident  between  the  aurelia 
and  caudatum  forms  do  not  owe  their  origin  to  the  difference  in  the 
mass  of  macronuclear  material.  On  a  priori  grounds  it  is  reasonable 
to  expect  the  more  rapid  multiplication  in  forms  with  the  greater 
proportion  of  micronuclear  material,  and  this  is  borne  out  in  the 
experiments  for  it  appears  from  the  curve  that  during  the  period 
of  abnormal  micronuclei,  when  the  smaller  amount  of  micronuclear 
material  was  distributed  in  two  nuclei,  the  organism  was  laboring 
under  abnormal  physiological  conditions  and  had  a  lower  division 
rate  than  when  the  normal  uninucleate  conditions  were  restored. 


lo  Gary  N.  Calkins 


EXPLANATION  OF  PLATE  i. 

M = macronu  cleus . 
m=micronucleus. 

Fig.  I. — Ex-conjugant  of  the  A  series  in  the  third  generation  after  union.  Four 
micronuclei  can  be  seen  to  the  right  of  the  macronucleus,  and,  in  addition,  a  rela- 
tively large  unabsorbed  fragment  of  the  old  macronucleus.  The  micronuclei  are 
precociously  divided  for  the  fourth  generation. 

Fig.  2. — Paramecium  aurelia  in  the  24th  generation.  The  two  micronuclei 
may  be  seen  at  the  lower  end  of  the  macronucleus,  one  on  each  side. 

Fig.  3. — Paramecium  aurelia  in  the  43d  generation.  The  two  micronuclei 
are  of  relatively  large  size,  and  are  dissociated  from  the  macronucleus  at  the  upper 
end  of  the  animal  (the  two  objects  at  the  right  of  the  macronucleus  are  foreign  par- 
ticles  deposited   on   the   organism). 

Fig.  4. — Paramecium  aurelia  in  the  46th  generation.  The  two  micronuclei 
are  at  the  upper  end  of  the  macronucleus,  and  cannot  be  seen  distinctly. 

Fig.    5. — Paramecium   aurelia   in   the   caudatum   phase    at    the    43d    generation 
after  union.    The  single  micronucleus  may  be  seen  at  the  extreme  right  end  of  the. 
nuclear  material.     It  is  noticeably  larger  than  the  micronuclei  of  the  aurelia  phase 
(compare  Fig.  2).     This  specimen  is  one  of  the  same  lot  as  that  represented  by  Fig.  3. 

Fig.  6. — Paramecium  aurelia  in  the  32d  generation  vdth  macronucleus  and 
micronuclei  in  division.  Three  of  the  micronuclei  are  shown  with  pointed  ends; 
the  fourth  is  out  of  focus  and  does  not  show. 

Fig.  7. — Paramecium  caudatum  phase  of  P.  aurelia,  in  the  220th  generation. 
The  relatively  large  micronucleus  is  dissociated  from  the  macronucleus.  This  soeci- 
men  was  killed  when  the  division  rate  was  high  (end  of  July;  cf.  curve). 


1         l^i     K     A.      M    J  i. 


:M. 


.  .  .  M 


w 


l-ic.  1. 


\^^^S^^^^^ 


M 


M 


.» 


Fig   2. 


Tk;.  3. 


IM...  .,. 


Fh-..  5- 


%        % 


Vu:.  6. 


I'lG-  y. 


THE  LIFE-HISTORY   OF  A   COCHLIDIAN    MOTH— .IDO- 
NETA  BICAUDATA  DYAR* 

Harrison    G.    Dyar, 

Custodian  of  Lepidoptera,  U.  S.  National  Museum,  Washington,  D.  C. 

The  larvae  of  the  Cochlididas,  or  slug  caterpillars,  are  especi- 
ally interesting  to  the  entomologist  on  account  of  their  peculiar 
forms  and  considerable  diversity  of  structure.  The  author  has 
been  interested  in  this  family  for  twenty  years,  and  has  been  so 
fortunate  as  to  work  out  the  life-histories  of  many  of  the  species 
inhabiting  the  Atlantic  coast  region  of  North  America,  as  well  as 
a  few  foreign  ones.  The  results  have  been  pubHshed  in  a  number 
of  articles  ;'"^^  so  it  is  not  necessary  to  repeat  here  the  general  struc- 
tural characters  of  these  larvae.  They  are  highly  specialized  forms 
of  Tineoidea  or  Microlepidoptera;  that  is,  the  larvae  are  highly 
specialized.  The  moths  have  not  shared  in  the  complication  of 
structure  exhibited  in  their  early  stages.  The  family  is  spread 
throughout  the  world,  and  is  doubtless  of  early  origin  in  geologic 
time. 

North  of  Florida  and  Texas,  where  peculiar  forms  occur,  26 
species  are  at  present  known.  Of  these  I  have  found  the  larvae 
of  20,  and  have  been  especially  interested  to  secure  the  remain- 
ing six,  if  possible.  It  was  the  search  for  one  of  these,  Monoleuca 
semijascia  Walker,  which  resulted  in  the  knowledge  of  the  life- 
history  of  Adoneta  hicaudata,  though  not  successful  in  its  immediate 
object.  In  1898  and  1899  I  made  collections  of  larvae  at  Morris 
Plains,  N.  J.,  where  it  was  said  that  Monoleuca  had  been  taken 
many  years  ago,  hoping  that  it  might  still  be  there.  The  larv£e 
were  very  small;  but  as  nothing  that  looked  to  be  the  one  sought 
appeared,  they  were  turned  over  to  Mr.  L.  H.  Joutel,  of  New  York. 
Later  in  the  season  Mr.  Joutel  called  my  attention  to  two  larvae 
which  were  new  to  him  that  he  had  noticed  among  his  stock,  and 
which  he  thought  were  from  among  those  I  had  given  him,  or  else 
came  from  Long  Island.  The  new  larva  was  allied  to  Adoneta 
spinuloides  Herrich-Schaeffer,  and  we  thought  it  might  be  the  Mono- 

♦Received  for  publication  October  5,   1905. 

II 


12  Harrison  G.  Dyar 

leuca,  since  that  genus  is  allied  to  Adoneta  as  adult  moths.  We 
were  not  then  successful  in  rearing  the  new  larva.  It  is  referred 
to  in  print^'*  as  Monoleuca  ( .?),  as  I  then  thought  it  might  be.  Sub- 
sequently, in  arranging  the  collections  at  the  National  Museum 
at  Washington,  I  found  an  inflated  larva  of  this  new  form,  together 
with  a  bred  female  adult,  prepared  by  Mr.  A.  Koebele,  formerly  of 
the  United  States  Department  of  Agriculture.  The  adult  was 
not  Monoleuca,  but  a  light-colored  form  of  Adoneta,  which  I  supposed 
must  be  Packard's  leucosigma,  though  I  could  not  be  sure  that  the 
new  larva  properly  belonged  to  it,  not  finding  Mr.  Koebele 's  notes. 
On  the  strength  of  this  specimen,  I  listed  leucosigma  as  a  variety 
of  spinuloides.^^  In  1903  and  1904  Mr.  W.  F.  Fiske,  of  the  Forestry 
Division  of  the  Bureau  of  Entomology,  was  collecting  at  Tryon, 
N.  C,  and  he  turned  up  Monoleuca  semijascia  in  numbers.  At 
my  request,  Mr.  Fiske  secured  eggs,  but  they  all  proved  infertile. 
In  the  fall  of  1904,  therefore,  I  went  to  Tryon  for  the  purpose  of 
finding  the  Monoleuca  larva.  I  was  unsuccessful;  but  in  the  search 
obtained  a  number  of  larvee  of  the  new  form  of  Adoneta.  In  the 
same  season  a  number  of  adults  were  collected  at  Plummer's  Island 
in  the  Potomac  River,  near  Washington,  by  Messrs.  August  Busck, 
H.  S.  Barber,  and  E.  A.  Schwarz,  and  it  became  evident  that  I 
had  before  me  an  undescribed  species  of  Adoneta,  which  was  not 
leucosigma  Packard.  I  therefore  published  a  description  of  it, 3° 
associating  with  it  the  new  larva. 

The  larvae  from  Tryon  resulted  in  several  male  and  one  female 
moths  in  July,  1905.  The  males  emerged  before  the  female,  and, 
in  flying  in  the  cage,  lost  their  legs,  so  that  they  quickly  destroyed 
themselves  by  buzzing  on  the  ground.  When  the  female  emerged, 
I  had  no  living  male  left.  I  placed  her  in  the  cage  at  Rock  Creek, 
near  Massachusetts  Avenue,  in  the  District,  but  no  male  was  at- 
tracted. The  next  night  Mr.  H.  S.  Barber  kindly  took  the  cage 
to  Plummer's  Island  and  remained  there  all  night,  but  again  with- 
out success.  However,  Mr.  Barber  took  at  light  two  males  of 
this  species,  which  he  kept  ahve  in  glass  tubes.  One  had  lost  its 
legs  and  was  useless,  but  the  other  was  in  good  condition  and  was 
placed  in  the  cage.  Mating  occurred  on  the  third  night  after  emer- 
gence of  the  female,  and  good  eggs  were  secured. 


Life-History  of  a  Cochlidian  Moth  13 

Adoneta  hicaiidata  is  remarkable  for  the  lateness  of  emergence 
of  the  adults,  late  July  and  early  August  being  the  time  of  flight. 
One  other  species,  Isochaeles  beutenmuelleri  Hy.  Edwards,  has 
similar  habits  and  a  similar  distribution.  Northern  New  Jersey 
and  Staten  Island  N.  Y.,^^  seem  to  be  the  limits  of  their  range 
northward,  and  it  is  doubtful  if  they  occur  so  far  north  in  every 
season.  Their  lives  as  larvae  are  as  long  as  those  of  any  other  species, 
namely  about  eight  weeks,  so  that  they  are  liable  to  be  destroyed 
by  early  frosts  before  maturing.  After  spinning  their  hard  cocoons 
on  the  ground  or  among  dead  leaves,  they  are  immune  to  cold, 
though  pupation  does  not  occur  till  the  following  spring.  The 
species  is,  of  course,  strictly  single-brooded. 

The  eggs  are  laid  in  groups  of  two  to  ten,  overlapping,  and  placed 
on  the  backs  of  the  leaves.  They  are  usually  deposited  in  low 
places,  young  red  oak  bushes  in  not  too  dense  woods  being  the  best 
situation.  None  were  found  on  the  higher  branches.  At  Tryon, 
in  1904,  all  the  larvae  taken  were  on  small  red  oak  trees.  In  1905 
I  took  a  larva  near  Washington  (Rosslyn,  Va,),  also  on  red  oak, 
and  a  number  at  Tryon  at  the  end  of  September  on  this  tree,  and 
also  on  other  plants,  white  oak  and  Ceanothus.  The  little  larvae, 
on  hatching,  separate,  having  no  tendency  to  be  gregarious.  Their 
first  business  is  to  molt,  which  they  do  within  two  days  of  hatching, 
without  having  fed  during  the  first  instar.  In  the  second  stage 
feeding  begins.  The  young  larvae,  up  to  the  fifth  or  sixth  stage, 
eat  the  lower  epidermis  and  parenchyma  of  the  leaf,  cutti  ig  short, 
scattered,  irregular  channels  of  about  the  width  of  their  bodies. 
They  frequently  change  their  feeding-place  and  pass  readily  from 
one  leaf  to  another,  but  of  course  cannot  leave  the  tree  on  which 
the  eggs  were  deposited.  When  large  enough,  they  begin  to  cat 
the  whole  leaf  from  the  end  or  side  inward,  as  the  other  species 
of  Cochlidians  do.  The  larvae  observed  by  me  passed  nine  stages, 
though  they  can,  no  doubt,  mature  in  eight  under  more  favorable 
conditions.  I  was  obliged  to  carry  my  larvae  to  northern  New 
York,  where  they  were  fed  on  yellow  birch,  oak  not  being  available, 
and  were  subjected  to  the  rigors  of  an  Adirondack  climate.  When 
matured  the  larvae  change  color  slightly  and  leave  the  tree  to  spin 
their  cocoons  among  dead  leaves  on  the  ground,  wherein  they  pass 
the  winter  in  the  prepupal  stage. 


14  Harrison  G.  Dyar 

As  compared  with  its  nearest  ally,  Adoneta  spinuloides  H.  S., 
the  larva  of  A.  hicaudata  is  narrower,  less  elevated  at  joint  5;  the 
horns  of  joint  13  are  long,  predominant  over  those  of  joint  12,  which 
exceed  those  of  joint  10;  the  widenings  of  the  dorsal  purple  band 
are  all  subequal,  five  widenings,  each  practically  alike,  no  pushing 
out  of  the  subdorsal  horns  of  joints  6  and  7;  caltropes  reduced 
on  the  anterior  side  horns,  joint  6,  et  seq.,  only  well  developed  on 
joints  12  and  13;  the  color  of  the  dorsal  band  is  bluer  purple  than 
spinuloides,   less   reddish   or   maroon. 

DESCRIPTION    OF    THE    SEVERAL     STAGES. 

Egg. — Elliptical,  colorless,  shining,  fiat,  the  surface  faintly  retic- 
ular. The  yolk  forms  a  slightly  opaque  central  mass,  leaving  a 
transparent  rim.  On  the  leaf  the  eggs  are  shining  and  transparent, 
resembling  drops  of  moisture.  Their  extreme  flatness  enables 
them  to  be  laid  overlapping  hke  shingles.  Size,  i. 4X0. 8X0.  i  mm. 
The  development  of  the  embryo  can  be  easily  observed  in  eggs 
laid  on  glass.  The  shell  is  thin  and  transparent,  merely  a  delicate 
skin.  It  is  impossible  to  detach  the  eggs  without  destroying  them. 
They  hatch  in  10  days. 

Stage  I. — Elliptical,  the  dorsum  flattened,  the  sides  oblique, 
the  venter  fiat.  Head  small  and  weak,  without  hard  chitinous 
covering;  mandibles  and  palpi  present,  but  reduced  and  weak; 
the  mandibles  with  four  equal  blunt  teeth  incapable  of  feeding, 
colorless.  A  subdorsal  and  a  lateral  row  of  processes  or  "horns," 
one  on  a  segment,  the  subdorsals  on  joints  3-13,  the  laterals  on 
joints  3,  4,  and  6-12.  The  subdorsals  of  joints  3,  4,  5,  8,  11,  12, 
and  13  are  large,  the  others  small;  the  laterals  of  joints  3  and  4 
are  large,  the  others  smaller,  but  not  as  small  as  the  small  subdorsals. 
Each  horn  bears  three  setas,  occasionally  but  two,  slender,  slightly 
clavate-tipped,  smooth.  The  setae  of  joint  2  are  not  elevated  on 
horns.  Skin  smooth.  Colorless,  whitish,  without  markings.  On 
hatching,  the  larvae  gathered  in  groups  as  they  had  been  laid, 
and  molted  in  two  days.     Length,  0.9  mm. 

Stage  II. — Head  rounded,  white,  clypeus  highly  triangular, 
reaching  about  half-way  to  the  vertex;  eyes  black;  mandibles 
stout,   brown-tipped.     Elliptical,    narrowed  behind,   dorsum  nearly 


Life-History  of  a  Cochlidian  Moth  15 

level.  Subdorsal  horns  of  joints  3-5,  8,  11,  12  spherical,  large, 
densely  spined,  those  of  joints  6,  7,  9,  and  10  minute,  with  one  tubercle 
and  one  spine  only,  and  each  approximated  to  the  adjoining  large 
horn;  subdorsal  horn  of  joint  13  small,  with  two  or  three  spines. 
Lateral  horns  alike  with  three  or  four  spines,  except  those  of  joints 
3  and  4,  which  are  larger  and  more  rounded.  Skin  rather  densely 
granular,  smooth,  without  markings.  The  spines  are  large,  with 
large  tubercular  bases  and  black  tips.  The  detachable  tip  is  nearly 
as  long  as  the  shaft  of  the  spine  and  is  not  enlarged  near  the  junction. 
These  spines  are  presumably  of  the  urticating  type,  as  they  have 
that  structure,  though  they  are  probably  too  small  to  pierce  the 
human  skin,  at  least  in  this  early  stage.  There  is  a  row  of  fine 
hairs  on  the  anterior  edge  of  the  hood  (joint  2).  Length,  0.9- 
1 .6  mm. 

Slage  III. — Head  elongate,  higher  than  wide,  whitish,  the  eyes 
in  a  round  black  spot,  the  mandible  brown-tipped.  Elongate  ellip- 
tical, flattened,  rounded  squarish,  narrowed  behind  more  than  in 
front,  the  dorsum  flat.  Subdorsal  horns  large,  rounded,  well  spined, 
those  of  joints  6,  7,  9,  and  10  represented  by  four  or  five  spines  on 
the  skin,  approximated  to  the  neighboring  horns.  Lateral  horns 
moderate,  rounded,  all  elevated.  Skin  granular  shagreened,  the 
granules  sharply  conical  over  the  dorsal  region  and  separated  by 
their  own  diameters  or  more,  on  the  sides  more  flattened  and  irreg- 
ular till  along  the  subventral  edge  they  form  a  pattern  resembling 
alligator  skin.  All  whitish  green,  no  marks.  The  depressed  spaces 
(i)  are  faintly  indicated,  paired.  The  cores  of  the  large  subdorsal 
horns  are  slightly  whitish.  The  spines  on  the  horns  have  the  detach- 
able tips  relatively  shorter  than  before,  being  not  over  one-third 
of  the  whole  spine  for  the  subdorsal  horns.  The  lateral  ones  are 
less  advanced,  and  have  the  structure  of  the  subdorsals  of  the  pre- 
vious stage.  Length,  i .  6-1 . 8  mm.  This  is  apparently  the  inter- 
polated stage. 

Stage  IV. — Elongate,  flattened,  tapered  behind,  the  ends  trun- 
cate; the  larva  generally  sits  a  little  curved.  Subdorsal  horns 
rounded,  large,  subequal  on  joints  3,  4,  5,  8,  11,  and  12,  the  pair 
on  joint  13  small,  not  as  yet  produced.  The  short  horns  of  joints 
6,  7,  9,  and  10  rounded,  well  spined,  not  so  strongly  appro .ximated 


1 6  Harrison  G.  Dyar 

to  the  neighboring  horns  as  before.  Lateral  horns  small,  sub- 
equal.  Skin  densely  granular  shagreened.  Depressed  spaces  (i) 
distinct,  paired,  a  little  whitish.  Translucent  greenish,  a  narrow 
white  hne  along  the  subdorsal  ridge  the  whole  length.  Horns 
slightly  yellowish-tinted,  with  a  trace  of  vinous  shading  at  the  ends 
of  the  body.  Later  there  appear  more  decided  traces  of  color. 
A  yellowish-white  band  in  the  subdorsal  ridge  is  broken  into  six 
slightly  obhque  broad  bars  under  the  long  horns,  elsewhere  faint; 
there  is  a  fine  hnear  straight  white  dorsal  line,  broken,  most  distinct 
centrally,  dividing  the  small  white  dots  of  depressed  spaces  (i). 
A  slight  purplish  infiltration  in  the  dorsal  space  between  these 
marks.     Length,  1.8-2.8  mm. 

Stage  V. — Elongate,  flattened,  tapered  behind,  the  subdorsal 
horn  of  joint  13  about  two-thirds  as  long  as  that  of  joint  12,  the 
rest  subequal,  those  of  joints  6,  7,  9,  and  10  very  small.  Lateral 
horns  of  joints  3  and  4  moderate,  the  rest  small.  Green,  whitish 
along  the  sides;  subdorsal  horns  yellow  with  a  yellowish- white 
subdorsal  band  below  them,  narrowed  in  the  spaces  between  to 
border  the  dorsal  space,  which  is  especially  elliptically  widened 
at  the  short  horns  of  joints  6-7  and  9-10.  A  broken  white  dorsal 
line;  depressed  dots  (i)  greenish,  impressed.  A  slight  purple 
infiltration  all  along  the  dorsal  space  between  the  markings.  Length, 
2 . 8-4 . 1  mm. 

Stage  VI. — Horns  of  joint  13  distinctly  elongated  into  tails> 
longer  than  the  subdorsals  of  joint  12.  EUiptical,  the  sides  wide 
centrally  and  flattened,  the  dorsum  long,  straight,  narrow.  Horns 
moderate,  the  short  subdorsals  distinct,  separate,  about  as  large 
as  the  lateral  horns,  of  which  those  of  joints  3  and  4  are  stouter, 
but  hardly  longer  than  the  rest.  Green,  the  subdorsal  ridge  broadly 
yellow,  narrowed  at  the  short  horns,  causing  the  dorsal  space  to 
expand  there,  apparently.  A  broken  white  dorsal  line  touching 
the  white  dots  of  depressed  spaces  (i).  Dorsal  space  partly  purple- 
filled.  A  broken  yellow  hne  beneath  the  lateral  horns.  Subdorsal 
horn  of  joint  3  slightly  red-tipped.  Spines  black-tipped;  skin 
densely  frosty  granular;  the  lateral  depressed  spaces  show  faintly  as 
large,  concolorous,  kidney-shaped  hollows.  Later  the  markings 
become  more  pronounced.     Green,  the  subdorsal  horns  of  joint  3 


Life-History  of  a  Cochlidian  Moth  17 

red,  those  of  joints  4  and  5  red-tipped,  the  rest  green.  Dorsal 
marking  expanded  in  the  intersegments  3-4,  4-5,  5-8,  8-10,  and 
very  shghtly  lo-ii  and  11-12,  the  first  and  last  green-filled,  the  rest 
dark  purple,  cut  by  the  yellow  dorsal  line  and  the  pale  depressed 
spaces  (i),  broken  at  joint  8,  broadly  yellow-edged  to  the  sub- 
dorsal horns.  Horns  with  stinging  spines,  no  caltropes.  Skin 
sparsely  clear  granular.     Length,  4.1-5.2  mm. 

Stage  VII. — As  before.  The  subdorsal  horns  of  joint  13 
are  long  and  tapered.  The  subdorsals  of  joints  3-5  are  red,  those 
of  12  and  13  sHghtly  red-tipped;  a  greener  fine  within  subdorsal 
ridge.     Length,   5.2-6.9  mm. 

Stage  VIII. — Subdorsal  horn  of  joint  3  a  little  longer  than  those 
of  joints  4  and  5,  but  all  now  very  short,  those  of  joints  6-11  still 
shorter,  of  12  a  little  longer,  of  13  long  and  tail-Hke.  Side  horns 
all  very  short.  The  level  narrow  dorsum  is  yellow  and  opaque,  the 
dorsal  band  of  purple  widens  into  five  subequal  patches,  nearly 
divided  at  joint  8,  where  the  purple  is  replaced  by  red  and  is  Unear 
only.  The  last  patch,  on  joints  11  and  12,  is  a  Uttle  smaller;  joint 
13  dorsally  green.  Horns  bright  red.  Sides  all  clear  green;  a 
broken  white  line  along  the  lateral  ridge  and  irregular  white  marks 
in  the  lateral  depressed  spaces.  Skin  granular;  spines  pale;  no 
caltropes,  except  perhaps  two  or  three  on  the  basal  anterior  green 
spot  of  the  horn  of  joint  13.  Dorsal  impressed  dots  (i)  greenish 
with  white  rings.  A  dorsal  white  line  narrowly  cuts  the  purple. 
Length,   6 . 9-9 . 3  mm. 

Stage  IX. — Long,  rather  narrow,  quadrate,  a  little  tapering 
behind.  Dorsum  broad,  flat,  not  arched  and  scarcely  higher  at 
joint  5,  yet  a  little  so.  Subdorsal  ridge  indicated  by  change  in 
direction.  Sides  perpendicular  or  nearly  so,  the  lateral  space  broad, 
continuous  with  the  subventral  space  which  is  infolded  in  the  middle. 
Subdorsal  horns  distinct,  short,  those  of  joints  3,  4,  5,  and  12  mod- 
erate, those  of  joint  13  long,  nearly  three  times  as  long  as  the  ones 
on  joint  12,  the  rest  short,  those  of  joints  8  and  11  a  Httle  larger 
than  the  others.  Side  horns  short,  sessile,  wider  than  long,  those 
of  joints  3  and  4  a  little  longer  than  those  of  6-12.  Caltrope 
patches  on  the  horns  of  joints  6-12  and  on  the  base  of  the  subdorsal 
horn  of  joint  13,  large  on  joints  12  and  13,  then  progressively  smaller 


1 8  Harrison  G.  Dyar 

till  the  horns  of  joints  6  and  7  have  only  a  few  or  no  caltropes.  Skin 
finely  clear  granular  except  on  the  horns.  No  end  spines.  Dorsum 
yellow-  or  red-shaded,  a  purple  band  with  white  glandular  dots 
and  central  dorsal  hne  much  as  in  spinuloides,  but  of  different  shape. 
It  widens  between  joints  3  and  4,  4  and  5,  then  moderately  widens 
on  joints  6  and  7,  narrows  to  a  sMght  bordering  of  the  white  dorsal 
line  over  joint  8,  widens  behind  the  horns  on  9  and  10,  moderately 
widens  between  joints  11  and  12  and  ends,  joint  13  being  green 
above.  A  bright  red,  diffuse,  subdorsal  band;  all  the  subdorsal 
horns  red.  Below,  a  yellow  stripe,  narrowly  red-edged,  waved. 
Sides  green,  a  row  of  yellow  dashes  along  the  lateral  horns,  green- 
edged  above;  yellow  rings  on  spaces  (4).  A  white  Hne  along  the 
subventral  edge.  Stinging  spines  short,  not  numerous.  Depressed 
spaces  (i)  and  (2)  represented  by  white  dots,  (i)  paired  and  on 
joints  3-4  and  4-5  also  double;  depressed  space  (4)  reniform,  dis- 
tinct; sHght  hollows  subventrally ;  spiracle  of  joint  5  moved  up 
out  of  Hne.     Length,  9.3-13  mm. 

Cocoon. — Nearly  spherical,  hard,  brown,  one  end  opening  Hke 
a  circular  lid  on  emergence,  though  there  is  no  sign  externally  of 
the  crevice  of  the  lid.  Spun  with  a  slight  veil  against  a  leaf.  Diam- 
eter, 5  mm;  length,  7  mm. 

Pupa. — With  the  characters  of  the  family.  DeHcate,  thin- 
skinned  with  the  members  free.  It  emerges  nearly  out  of  the  cocoon 
when  the  adult  issues. 

Food  plants. — The  red  oak  is  the  preferred  plant,  though  the 
larvae  will  feed  on  almost  any  smooth-leaved  tree.  I  found  one 
on  maple  at  Tryon,  the  others  mostly  on  oak.  In  confinement  my 
larvae  fed  on  yellow  birch,  which  they  seemed  to  prefer  to  soft  maple. 

REFERENCES. 


Dyar  AND  Morton.     General  account,  Jour.  N.  Y.  Ent.  Soc,  1895,  3,  pp.  145-51. 

"       "  "  Apoda  y-inversa  Pack.,  ibid.,  1895,  3,  pp.  151-57. 

"       "  "  Sibine  stimulea  Clem.,  ibid.,  1896,  4,  pp.  1-9. 

Dyar.  Euclea  delphinii  Boisd.,  Florida  Form,  ibid.,   1896,  4,  pp.  125-29. 

"        Tortricidia  pallida  H.-S.,  ibid.,   1896,  4,   pp.  167-72. 

"        Eulimacodes  scapha  Harr.,  ibid.,  1896,  4,  pp.  172-78. 

"        Phobetron  pithecium  Sm.  &  Abb.,  ibid.,  1896,  4,  pp.  178-84. 

"        Sisyrosea  textula  H.-S.,  ibid.,   1896,  4,   pp.  185-90. 

"        Tortricidia  fasciola  H.-S.,  ibid.,  1897,  5,  pp.  1-5. 


Fig.   I. 


""'"■''■  ■'■'^ 


Fig. 


_T-rf^^-^^'--''>'-  "••-•^ 


■.■  ■„.,■. 11 1- '.  ■i.'i-..,i^-nT^ 


Fig.  6. 


Fig.  2. 


Fig.   7. 


Fig.  4. 


-snr-"v-v :'.'.";'   ;:,, ';.'.; ju.  M^S3 


Fig.  5. 


Life-History  of  a  Cochlidian  Moth  19 

10.  Dyar.     Adoneta  spinidoides  H.-S.,  ibid.,  1897,  5,  pp.  5-10. 

11.  "  Enclea  indetermina  Boisd.,  ibid.,  1897,  5,  pp.  10-14. 

12.  "  Euclea  delphinii  Boisd.,  ibid.,  1897,  5,  pp.  57-61. 

13.  "  Parasa  chloris  H.-S.,  ibid.,  1897,  5,  pp.  61-66. 

14.  "  Calybia  slossoniae  Pack.,  ibid.,  1897,  5,  pp.  121-26;  1898,  6,  pp.  158-60. 

15.  "  Apoda  biguttata  Pack.,  ibid.,  1897,  5,  pp.  167-70. 

16.  "  Packardia  geminata  Pack.,  ibid.,  1898,  6,  pp.  1-5. 

17.  "  Packardia  elegans  Pack.,  ibid.,  1898,  6,  pp.  5-9. 

18.  "  Heterogenea  fle.xuosa  Grote,  ibid.,   1898,  6,  pp.  94-98. 

19.  "  Tortricidia  testacea  Pack.,  ibid.,  1898,  6,  pp.  151-55. 

20.  "  Heterogenea  shurtleffii  Pack.,  ibid.,  1898,  6,  pp.  241-46, 

21.  "  Natada  nasoni  Grote.,  ibid.,  1899,  7,  pp.  61-67. 

22.  "  Cochlidion  avellana  Linn.,  ibid.,  1899,  7,  pp.  202-08. 

23.  "  Summary  and  Conclusion,  ibid.,  1899,  7,  pp.  234-53. 

24.  "  Note  on  Doubtful  larva,  ibid.,  1899,  7,  236  note. 

25.  "  Sibine  jusca  Stoll,  Ent.  News,  1900,  11,  pp.  517-26. 

26.  "  Isochaetes  heutenmuelleri  Hy.  Edw.,  Proc.  Ent.  Soc.  Wash.,  1901,  4,  p.  300. 

27.  JouTEL.  Note  on  Occurrence,  Jour.  N.   Y.  Ent.  Soc,  1902,  10,  p.  248. 

28.  Dyar.     Catalogue  of  species.  Bull.  52,  U.  S.  N.  M.,  1903,  pp.  354-58. 

29.  "  Synonymy,  Bull.  52,  U.  S.  N.  M.,  1903,  p.  355,  No.  4085a. 

30.  "  Description  of  new  species,  Jour.  N.  Y.  Ent.  Soc,  1904,  12,  p.  43. 

31.  "  Catalogue  of  species,  Proc.  U.  S.  N.  M.,  1905,  29,  pp.  359-96. 

EXPLANATION  OF  PLATE  2. 

Fig.  I. — The   larva  in   Stage   I,   dorsal   view. 

Fig.  2. — One  of  the  subdorsal  horns  of  Stage  I,  the  spines  of  the  next  stage  appear- 
ing by  transparency.     The  larva  was  about  to  molt. 
Fig.  3. — A  spine  of  Stage  II. 
Fig.  4. — The  mature  larva  in  position  of  feeding. 
Fig.  5. — A  seta  of  the  last  stage. 
Fig.  6. — An  urticating  spine  of  the  last  stage. 
Fig.  7. — A  caltrope  spine  of  the  last  stage. 


EXPERIMENTAL    METHODS   AS   APPLIED   TO   WATER- 
AND    SEWAGE-WORKS    FOR    LARGE    COM- 
MUNITIES* 

George  W.  Fuller. 

That  progress  means  advance  in  knowledge  and  the  gradual 
transition  of  information  from  the  unknown  to  the  known  is  of 
course  a  truism.  The  manner  by  which  progress  is  made  in  different 
lines  of  work  varies  widely.  The  experimental  method  as  appHed 
to  the  teaching  of  science  in  educational  institutions  has  been  of 
great  benefit,  and  the  application  in  a  more  systematic  manner 
than  formerly  of  the  so-called  "cut  and  try"  method  of  the  early 
inventors  has  resulted  in  more  substantial  progress  in  many  mechan- 
ical and  industrial  Hues.  By  the  man  of  affairs  this  advance  in 
knowledge  is  referred  to  as  additional  experience.  It  is  not  the 
purpose  of  this  paper  to  attempt  an  analysis  of  the  manner  by 
which  knowledge  in  general  is  advanced,  but  to  refer  somewhat 
briefly  as  a  matter  of  record  to  the  way  in  which  there  have  been 
solved  various  sanitary  problems  which  a  quarter  of  a  century  ago, 
for  financial  or  other  reasons,  seemed  out  of  question. 

It  is  fitting  on  this  occasion  that  this  topic  should  be  touched 
upon,  owing  to  the  influence  exerted  upon  this  hne  of  work  by  the 
two  institutions  with  which  Professor  Sedgwick  has  been  most 
actively  connected  in  recent  years,  viz.,  the  Massachusetts  State 
Board  of  Health  and  the  Massachusetts  Institute  of  Technology. 
The  former  has  for  years  been  the  foremost  among  the  state  boards 
of  health  of  America  in  leading  local  authorities  to  improve  their 
water-  and  sewage- works  in  an  efficient  and  economical  manner. 
Its  influence  has  extended,  not  only  to  other  states,  but  also  to 
numerous  places  throughout  the  civilized  world.  The  Massachusetts 
Institute  of  Technology  has  exerted  a  less  direct  influence;  but,  as  a 
training  school  for  many  who  have  taken  a  prominent  part  in  improv- 
ing water-  and  sewage-works,  this  institution  and  a  number  of  its 

♦Received  for  publication  March  24,   1906. 

20 


Experimental  Methods  in  Water-  and  Sew  age- Works    21 


teaching  stafif  have  achieved  resuhs  which  will  be  more  fully  appre- 
ciated as  the  years  go  by. 

While  the  experimental  method  so  successfully  applied  in  the 
student  laboratory  may  in  its  way  call  for  just  as  conscientious  and 
dihgent  effort  as  when  applied  to  projects  involving  immense  sums 
of  money,  yet  the  responsibility  associated  with  the  latter  is  far 
greater.  It  is  necessary,  in  carrying  out  successfully  these  large 
practical  problems,  not  only  to  draw  correct  conclusions  from  full 
representative  premises  of  a  complicated  nature,  but  also  to  adjust 
the  project  to  a  reasonable  business  basis,  to  make  it  fairly  well 
understood  by  non-technical  officials  and  citizens,  and  to  defend 
it  from  the  obstructionists  who,  for  political  or  selfish  reasons,  cross 
the  pathway  of  nearly  every  large  public  enterprise.  In  meeting 
these  requirements  there  has  been  called  forth  a  series  of  efforts 
which  are  of  great  significance  to  the  public  from  the  sanitary  and 
financial  standpoints,  and  which  form  notable  achievements  in  the 
field  of  apphed  science. 

benefits  of  improved  sanitary  works. 

Improved  water-  and  sewage-works  of  course  do  not  explain 
by  any  means  the  entire  improvement  which  for  the  past  quarter 
of  a  century  has  been  so  characteristic  of  the  sanitary  conditions 
of  a  majority  of  the  civilized  communities  of  the  world.  But,  illus- 
trative of  the  scope  of  such  improvements  under  the  guidance  of 
wise  sanitary  authorities,  it  is  of  interest  to  point  out  the  markedly 
decreased  death-rates  in  Massachusetts  from  water-borne  diseases, 
of  which  typhoid  fever  is  the  typical,  but  not  the  only  one. 

Death   Rates  per   100,000  Population  from  Typhoid  Fever   in  the  State  of  Massachusetts 

BY  Five-Year  Periods  from  1881  to  Date. 


Period 

Rate 

Period 

Rate 

iSSi-iSSs  

41 
46 

34 

1896-1900 

igoi-1904 

26 

1886-1890  

1891-1895  

19 

What  is  true  of  Massachusetts  is  in  a  general  way  true  of  many 
sections  of  both  America  and  Europe  where  the  population  has 
become  quite  dense,  and  demands  for  water-  and  sewage-works  of 
satisfactory  character  have  pressed  forward   for  attention  in  recent 


22  George  W.  Fuller 

years.  No  attempt  will  be  made  here  to  show  the  great  sanitary 
benefit  derived  from  other  factors  than  improved  water  supply  and 
sewerage,  as  this  matter  is  clearly  set  forth  in  standard  works  upon 
sanitation  and  official  reports  from  various  quarters  of  health  author- 
ities who  deal  with  the  accomphshments  of  the  modern  health 
officers. 

It  is  sufficient  here  to  present  clearly  to  the  reader  the  thought 
that  modern  sanitary  science  has  greatly  increased  the  comfort  and 
safety  of  living.  In  various  cities  the  reductions  in  death-rates  have 
been  far  greater  than  as  given  above  for  the  state  of  Massachusetts. 
This  is  especially  true  in  cities  where  badly  polluted  water  supplies 
have  been  replaced  by  improved  supplies.  Numerous  instances 
of  this  sort  are  on  record  where  annual  typhoid  fever  deaths  of  50 
to  100  per  100,000  have  been  reduced  to  about  20.  These  cities 
include  not  only  those  now  receiving  upland  waters  from  unpolluted 
sources,  and  ground  water,  but  also  those  having  filtered  water 
from  earlier  but  polluted  sources;  for  example,  Lawrence,  Mass., 
Albany,  N.  Y,,  York,  Pa.,  and  Lorain,  Ohio.  Intestinal  diseases 
other  than  typhoid  fever  have  been  so  reduced  as  to  lower  the 
general  death-rate  materially.  For  low-lying  communities  like 
New  Orleans  modern  drainage  has  lessened  notably  the  general 
death-rate,  and  sewerage  brings  safety  as  well  as  comfort  to  com- 
munities, among  other  ways  by  ehminating  privy  vaults  and  the 
likelihood  of  disease  transmission  by  flies,  etc. 

As  to  financial  considerations,  it  is  difficult  to  present  the  full 
significance  of  this  feature  to  the  general  reader  without  statistics 
which  would  be  out  of  place  in  a  short  general  article  like  this. 
Usually  water-purification  projects  proper  cost  about  $3  to  $5  per 
inhabitant  served;  but  pumping,  force-mains,  conduits,  reservoirs, 
and  other  associated  appurtenances  sometimes  increase  the  invest- 
ment to  $15  to  $25  per  capita.  Sewage  purification  frequently 
is  more  expensive  than  water  purification.  Upon  capitahzing  the 
operating  expenses,  it  is  found  that  modern  sanitary  works,  while 
involving  only  small  costs  for  the  individual,  reach  sums  of  millions 
and  tens  of  miUions  of  dollars  for  our  large  cities.  The  solution 
of  these  problems  has  brought  many  added  duties  to  sanitary  author- 
ities, to  professional  men  who  assume  the  responsibihty  for  con- 


Experimental  Methods  in  Water-  and  Sewage-Works    23 

structing  and  operating  the  required  works,  and  to  the  educational 
institutions  which  give  technical  training  to  young  men  desirous 
of  entering  this  field  of  work. 

new  conditions  which  have  been  encountered. 

Twenty-five  years  ago  the  large  cities  of  America  were,  of  course, 
provided  with  public  water  supplies,  and  many  of  them  had  pro- 
gressed considerably  in  adopting  sewerage  systems,  although  these 
latter  now  appear  to  have  been  more  or  less  crude.  Sewage-purifica- 
tion plants  and  water-purification  plants,  with  perhaps  half  a  dozen 
small  and  scattered  exceptions,  were  practically  unknown.  A 
large  proportion  of  the  water  supplies,  especially  in  the  Central 
West,  were  seriously  objectionable  in  the  excessive  quantities  of 
mud  which  they  carried,  and  so  different  in  their  nature  from  the 
comparatively  simple  filtration  projects  of  Europe  that  engineers 
naturally  hesitated  for  financial  reasons  to  attempt  their  construction, 
to  say  nothing  of  the  question  whether  they  would  be  able  to  give 
satisfactory  service.  The  bacterial  and  hygienic  aspects  of  these 
problems,  which  are  now  recognized  to  be  of  so  much  importance, 
were  almost  unknown.  In  fact,  the  germ  theory  of  disease  had 
not  risen  to  general  acceptance. 

The  medical  man  interested  in  pubHc  health  knew  in  a  general 
theoretical  way  what  he  wanted,  but  he  was  ordinarily  unable  to 
state  his  requirements  in  a  manner  understood  by  the  engineer 
or  by  the  taxpayer.  Engineers  were  able  to  build  any  reasonable 
works,  but  were  unable  to  learn  in  terms  of  the  constructor  what 
was  required  with  sufficient  definiteness  to  allow  them  to  make  even 
preliminary  sketches  and  estimates  of  cost.  The  chemist  and 
bacteriologist  occupying  an  intermediate  position  produced  with 
ill-suited    methods  analytical  data  full  of    mystery  for  everybody. 

Misunderstanding  continued  until  men  interested  in  various 
lines  of  applied  sanitary  science  co-operated  in  a  manner  to  make 
themselves  mutually  understood.  The  successful  movement  to 
this  end,  at  least  in  the  United  States,  had  its  inception  chiefly  in 
Massachusetts  some  20  years  ago.  It  has  resulted  during  the  past 
dozen  years  in  some  notably  well-balanced  designs  for  American 
water-   and  sewage- works,   which   have   demonstrated   their  sound 


24  *  George  W.  Fuller 

merit  in  practice.     In  this  article  it  is  the  endeavor  to  outHne  the 
development  of  this  aspect  of  experimental  methods. 

experimental    methods    in    MASSACHUSETTS. 

The  experimental  methods  which  have  been  put  in  practice  in 
America  so  much  in  recent  years  may  be  defined  as  the  bringing- 
together  of  rehable  preliminary  data  from  the  engineering,  chemical, 
bacterial,  and  hygienic  standpoints,  in  order  that  efficient  sanitary 
works  may  be  built  for  a  wide  range  of  local  conditions  within  the 
limits  of  reasonable  cost ;  and  if  data  are  inadequate  for  fair  assump- 
tions, then  the  procuring  of  the  needed  data  by  practical  tests  on  a 
small  scale. 

It  is  the  Massachusetts  State  Board  of  Health  to  which  credit 
is  principally  due  for  developing  this  method  to  serve  as  a  guide 
for  such  works.  In  1886,  when  the  present  board  was  organized, 
one  of  its  first  steps  was  to  establish  the  Lawrence  Experiment 
Station,  whereby  data  were  to  be  secured  to  show  the  best  means 
available  under  various  local  conditions  for  purifying  water  and 
sewage.  The  legislature  enacted  that  this  board  should  serve  as 
a  sanitary  tribunal,  before  which  the  local  authorities  should  place 
their  projects  for  water-  and  sewage-works,  and  whose  approval 
was  requisite  before  the  state  authorities  granted  the  local  author- 
ities permission  to  issue  bonds  for  their  construction.  This  experi- 
ment station  has  been  in  continuous  service  since  the  autumn  of 
1887,  and  has  attained  a  high  reputation  among  various  workers 
in  the  field  of  sanitary  science  throughout  the  world,  in  addition 
to  fulfilling  its  main  purpose  of  aiding  the  citizens  of  Massachusetts 
in  economically  improving  their  public  works,  whereby  to  a  material 
degree  the  health  and  comfort  of  the  people  of  the  state  have  been 
enhanced.  These  results  are  so  well  known  that  it  is  needless 
here  to  go  into  detail. 

The  classical  investigations  at  Lawrence,  as  set  forth  in  the  annual 
reports  for  the  past  15  years,  have  undoubtedly  done  more  than 
any  other  series  of  investigations  in  the  world  to  place  the  science 
of  purifying  water  and  sewage  on  a  sound  practical  basis.  It  is 
true  that  earlier  workers  abroad  had  previously  taken  important  steps 
along  some  of  these  lines,  and  that  sand  filtration  of  water  had  for 


rROPERH  UBRART 

II,  C.  State  C«U«f« 


Experimental  Methods  in  Water-  and  Sewage-Works     25 

many  years  been  in  use.  But  they  did  not  secure  comparable  paral- 
lel data  from  the  engineering,  chemical,  and  bacterial  standpoints 
with  anything  like  the  completeness  obtained  at  Lawrence,  whereby 
the  laws  governing  successful  practice  could  be  broadly  stated  for 
a  wide  range  of  conditions. 

Not  only  has  the  Massachusetts  State  Board  of  Health  availed 
itself  of  a  testing  department,  but  with  other  departments  it  has 
placed  itself  in  a  position  to  utilize  such  data  advantageously.  This 
has  been  done  by  an  analytical  department  procuring  data  at  fre- 
quent intervals  as  to  the  character  of  various  water  supplies,  rivers, 
effluents,  etc.,  and,  more  especially,  by  a  well-trained  engineering 
corps  which  applies  the  various  data  to  the  needs  of  each  problem 
coming  to  the  attention  of  the  board. 

That  the  Massachusetts  State  Board  of  Health  handles  well 
the  work  coming  within  its  jurisdiction  is  conceded  by  all  in  a  posi- 
tion to  know  of  it  intimately.  It  is  true  that  the  board  is  criticised 
for  not  devoting  itself  more  enthusiastically  to  studies  of  methods 
finding  favor  elsewhere,  but  this  criticism  has  little  to  support  it. 
The  board  properly  confines  itself  to  the  solution  of  problems  within 
the  state,  and  of  course  does  not  consider  it  necessary  to  do  more 
than  keep  generally  familiar  with  other  methods,  no  matter  how 
suitable  they  may  be  for  work  elsewhere. 

EXPERIMENTAL    METHODS    ELSEWHERE    IN    AMERICA. 

In  water  purification  the  Massachusetts  problems  arc,  generally 
speaking,  much  easier  and  simpler  than  those  of  the  Central  West 
and  South,  where  enormous  quantities  of  silt  and  clay  complicate 
the  necessary  works  for  treating  the  water,  and  add  materially  to 
the  cost  as  regards  both  construction  and  operation.  In  a  manner 
similar  to  the  procedure  at  Lawrence,  these  problems  were  worked 
out  at  LtDuisyille,^  Pittsburg,  Cincinnati,  and  New  Orleans.  At 
numerous  other  places  the  experimental  method  has  been  used 
in  adapting  more  strictly  the  design  of  works  to  local  conditions, 
especially  in  the  preliminary  treatment  of  turbid  waters  (Phila-  V 
delphia  and  Harrisburg),  the  removal  of  color  from  surface  waters 
(Providence),  of  iron  from  ground  waters  (West  Superior),  the 
softening  of  hard  waters   (Columbus),  and  the  removal  of  tastes 

X 


\ 


26 


George  W.  Fuller 


\/ 


and  odors  (Reading  and  Springfield).  These  problems  have  all 
been  carefully  studied  in  small  test  devices  for  securing  data  neces- 
sary for  advantageous  design  and  operation. 

Sewage  purification  has  also  been  studied  under  various  local 
conditions  at  several  places,  especially  at  Worcester,  Mass.;  Paw- 
tucket,  R.  I.;  Berlin,  Ont.;  the  Institute  of  Technology,  Boston; 
Columbus,  Ohio;  and  Waterbury,  Conn.  In  most  cases  the  sewage 
studies  have  arisen  because  of  inability  or  great  expense  in  applying 
the  well-known  Massachusetts  method  of  intermittent  filtration 
through  sand,  or  because  of  peculiarities  of  the  local  sewage. 

A  partial  hst  of  the  more  prominent  investigations  as  to  purifying 
water  and  sewage,  with  dates  and  approximate  costs,  is  as  follows: 

List  of  Special  Investigations  on  Water  and  Sewage  Purification. 


Place 


Lawrence,  Mass 

Pro\ndence,  R.I 

Louiswlle,  Ky 

Reading,  Pa 

Pittsburgh,  Pa 

Cincinnati,  Ohio 

West  Superior,  Wis 

Washington,  D.  C 

Richmond,  Va 

New  Orleans,  La \. 

Worcester,  Mass. .  .  .'l.V^O-.'. 

Philadelphia,  Pa 

Springfield,  Mass 

Harrisburg,  Pa 

Massachusetts  Institute  of  Technology,  Boston. 

Columbus,  Ohio 

Waterburv,   Conn 

Total 


Date 


1887  to  date 
1893-94 
1895-97 

1897 
1897-98 
I 898-99 
1898-99 

1899-1900 

1900 

1900-1 

1900  to  Mate 
1900-5 
1901-3 
1903-4 

1903  to  date 
1904-S 

1905  to  date 


Work 


Water  and  sewage 
Water 


Sewage 
Water 


Sewage 

Sewage  and  water 

Sewage 


Approximate 
Cost 


$175,000 
5.000 

47-395 
I,SOO 

36,286 

41,588 

2,000 

8,000 

2,000 

23,606 

37,000 

172,000 

18,000 

25,000 

20,000 

44,004 

10,000 

668,379 


It  is  not  pretended  that  the  above  list  is  complete.  In  fact, 
there  are  other  tests  which,  while  small  and  of  short  duration,  have 
had  much  to  do  with  professional  opinion.  Perhaps  the  most 
important  were  demonstrations  at  Louisville  and  St.  Louis,  many 
years  ago,  that  plain  sand  filtration  was  incapable  of  treating  the 
muddy  Ohio  and  Mississippi  River  waters  after  plain  sedimenta- 
tion in  large  basins. 

The  benefit  derived  from  the  experience  of  the  owners  of  propri- 
etary devices  cannot  be  overlooked — especially  in  regard  to  various 
appliances  of  mechanical  filters  which  occasioned  the  expenditure 
of  much  money  before  being  brought  to  their  present  state  of  devel- 
opment.    At  Louisville  alone  the  five  competing  filter  companies 


Experimental  Methods  in  Water-  and  Sewage-Works    27 

spent  more  than  $50,000.     More  recently  their  devices,  when  tested, 
have  been  purchased  at  the  beginning. 

While  this  paper  is  devoted  essentially  to  methods  of  purifying 
water  and  sewage  by  works  partly  or  wholly  of  artificial  construc- 
tion, mention  should  be  made  of  the  important  advances  in  the 
allied  lield  of  water  supply  from  storage  reservoirs,  and  the  disposal 
of  sewage  by  dilution.  Among  the  more  prominent  investigations 
of  this  kind  should  be  stated  those  field  surveys  and  laboratory 
studies  made  in  connection  with  the  Chicago  Drainage  Canal,  the 
additional  water  supply  of  New  York  City,  the  improvement  of 
the  Charles,  Mystic,  and  Neponset  Rivers  in  Massachusetts,  and 
foreshore  pollution  along  the  Massachusetts  coast.  Several  hundred 
thousands  of  dollars  have  been  expended  on  these  investigations. 

object  and  advantages  of  experimental  methods. 

The  purposes  of  applying  experimental  methods  to  problems 
of  water  and  sewage  purification  are  chiefly  threefold,  as  follows: 

1.  To  provide  data  for  the  official  and  technical  authorities, 
to  enable  them  to  adapt  new  works  most  advantageously  to  the 
local  conditions,  and  to  indicate  dimensions  and  other  physical 
conditions  permitting  contract  plans  to  be  prepared  and  the  cost 
of  construction  to  be  approximately  estimated. 

2.  To  educate  the  non-technical  public,  who  as  citizens  and 
taxpayers  are  interested  in  public  works. 

3.  To  provide  data  so  that  the  officials  can  operate  effectively 
the  works  after  they  are  completed,  and  forecast  the  approximate 
cost  of  operation. 

Technical  data. — In  regard  to  the  first  object  accompHshed, 
that  of  enabling  city  officials  and  their  technical  advisers  to  design 
economically  works  of  a  suitable  character,  it  goes  without  saying 
that  this  has  been  of  the  greatest  importance,  and  is  a  strong  factor 
in  explaining  the  rapid  strides  in  successful  sanitary  works  accom- 
plished during  the  past  few  years.  It  has  frequently  been  the  advice 
of  technical  men,  in  dealing  with  problems  which  differ  from  those 
successfully  solved  elsewhere,  to  make  tests  for  a  year  or  so  at  a 
cost  approximating  the  interest  for  one  year  for  the  works  contem- 
plated.    In   this  way  the  cost  of  errors  and  unbalanced  designs 


28  George  W.  Fuller 

has  been  largely  minimized,  and  the  efficiency  has  been  increased. 
In  the  field  of  water  and  sewage  purification  the  information  and 
experience  now  available  are  sufficient  in  a  majority  of  cases  to 
enable  these  problems  to  be  advantageously  handled  by  experienced 
workers  along  these  lines.  There  are  some  problems,  however, 
that  still  can  be  advantageously  treated  by  the  experimental  method. 
They  refer  especially  to  sewage  problems  in  which  trade  wastes 
are  involved,  and  to  water  problems  where  the  composition  of  the 
water  is  quite  unusual  in  some  particulars  on  frequent  occasions. 
From  the  technical  standpoint,  however,  the  field  of  water  and 
sewage  purification,  broadly  speaking,  has  passed  beyond  the  experi- 
mental stage,  and  the  advances,  both  as  to  efficiency  and  economy, 
are  largely  to  be  gained,  not  from  experimental  plants,  but  by  the 
careful  and  more  systematic  operation  of  works  in  practice.  Such 
studies  will,  of  course,  lead  to  improvements  which  can  be  taken 
advantage  of  in  the  construction  of  new  works,  and  will  gradually 
bring  to  a  higher  plane  of  excellence  the  art  of  water  and  sewage 
purification  on  its  present  scientific  basis. 

Educational  aspect. — It  is  frequently  said  that  communities 
progress  in  proportion  to  the  advance  in  knowledge  of  the  average 
citizen,  or  to  the  mean  knowledge  of  the  community  as  a  whole. 
There  is  a  good  deal  in  this,  and  it  brings  forcibly  to  mind  the  neces- 
sity of  educating  the  pubhc  as  to  what  improved  sanitary  conditions 
really  mean,  and  of  letting  them  ascertain  for  themselves  what  can 
be  accompHshed  along  these  hnes  in  the  field  of  applied  science. 
Non-technical  people  have  a  natural  aversion  to  the  word  "experi- 
ment," notwithstanding  the  aid  derived  from  devices  which  not 
improperly  may  be  termed  experimental.  While  the  term  "experi- 
ment station"  from  its  use  at  Lawrence  and  a  few  other  places 
seems  to  have  a  firm  footing  in  some  localities,  it  is  gradually  giving 
place  to  the  term  "testing  station."  This  is  a  much  preferable 
expression  in  many  ways,  as  it  disarms  the  criticism  of  many  who 
seem  to  think  that  these  investigations  are  conducted  in  a  "hit  or 
miss"  manner,  much  after  the  fashion  of  the  early  inventors.  This 
is  not  so,  as  experimental  methods,  as  now  ordinarily  apphed  to 
water-  and  sewage-works,  are  aimed  at  testing  procedures  found 
successful  elsewhere,   but  which  may  require  adaptation   to  local 


Experimental  Methods  in  Water-  and  Sewage-Works    29 

conditions  in  regard  to  some  details.  Their  magnitude,  while  rela- 
tively small  for  reasons  of  economy,  is  still  much  greater  than  that 
which  seems  to  be  taken  for  granted  by  numerous  citizens,  who 
associate  the  word  "experiment"  with  a  test  tube,  or  with  a  mechani- 
cal device  which  is  so  imperfect  that  no  one  dares  to  build  it  on 
a  large  scale  without  further  experiments.  The  methods  of  puri- 
fying water  and  sewage  have  now  advanced  to  a  degree  where  the 
phrase  "testing  station"  in  new  projects  will  unquestionably  displace 
"experiment  station;"  and  the  testing  of  these  processes  where 
unusual  conditions  are  expected  will  assume  a  dignity  comparable 
with  that  of  the  regular  departments  which  systematically  test 
cement,  steel,  and  other  materials  used  for  building  purposes.  In 
fact,  it  is  interesting  to  note  that  the  laboratories  at  many  testing 
stations  have  been  utilized  regularly  for  testing  construction  materials. 

Where  water-  and  sewage-purification  projects  involve  hundreds 
of  thousands  of  dollars  or  more  for  construction  costs,  the  so-called 
experimental  methods,  as  applied  in  accordance  with  the  foregoing 
statements,  have  given  wonderful  courage  in  many  places  to  offi- 
cials who  otherwise  would  very  naturally  have  been  in  a  hesitating 
frame  of  mind,  and  inclined  more  to  listen  to  the  "doubting  Thom- 
ases" who  in  all  communities,  for  selfish  or  other  reasons,  appear 
as  opponents  and  obstructionists  to  modern  sanitary  works.  Even 
if  the  technical  advisers  of  the  projects  were  not  assisted  by  such 
data,  it  is  quite  likely  that  the  testing  station  for  many  projects 
would  indirectly  in  this  way  do  far  more  good  than  the  cost  involved, 
in  saving  lives  and  in  hastening  the  day  when  communities  will 
meet  their  problems  in  accordance  with  the  best  information  available. 

In  speaking  of  the  educational  benefit  derived  from  applying 
experimental  methods  to  water-  and  sewage-works,  the  technical 
men,  especially  those  in  charge  of  the  tests,  have  an  important  duty 
to  perform  in  teaching  non-technical  officials,  and  various  citizens 
who  are  interested  in  the  work,  the  fundamental  principles  of  the 
process  involved,  and  in  assisting  them  in  ascertaining  what  prac- 
tical works  would  mean,  both  hygienically  and  financially.  Along 
this  general  line  the  Institute  of  Technology  has  played  an  impor- 
tant role,  largely  through  having  had  for  many  years  on  its  teaching 
stafi"  a  man  who  to  an  extraordinary  degree  possesses  the  faculty 


30  ,  George  W.  Fuller 

of  getting  fundamental  truths  of  sanitary  science  before  his  hearers 
in  such  an  attractive  manner  that  they  never  forget  them.  It  is 
the  behef  of  the  writer  that  the  vi^ork  accomplished  by  Professor 
Sedgwick  along  this  line  is  unequaled  by  that  of  any  other  man 
in  this  country,  either  in  educational  or  other  lines,  and  that  this 
fact  in  a  few  years  will  be  far  more  widely  realized  than  at  present, 
when  his  younger  pupils  throughout  the  country  reach  an  age  where 
their  work  will  be  felt  in  the  communities  in  which  they  live.  This 
influence  is  already  to  be  found  in  many  unexpected  places,  and 
forms  a  wonderful  tribute  to  the  success  accomplished  by  Professor 
Sedgwick  in  one  of  his  many  lines  of  usefulness. 

Operation  oj  works. — After  water-and  sewage-purification  works 
are  constructed,  it  is  imperative  that  they  shall  be  operated  in  an 
intelligent  and  efficient  manner.  The  benefit  of  this  has  long  been 
demonstrated  in  Europe,  and  the  absence  of  such  supervision  in 
many  places  in  America  shows  the  folly  of  careless  and  indifferent 
management.  No  matter  how  well  water-  and  sewage-purification 
works  may  be  designed  and  built,  there  is  no  engineer  who  can  give 
assurance  that  the  results  accomplished  will  be  satisfactory  unless 
the  works  are  well  managed.  Not  only  must  the  works  produce 
a  result  which  is  satisfactory  from  a  scientific  standpoint,  but  their 
behavior  should  be  put  before  the  citizens  in  a  way  that  will  inspire 
confidence.  When  fair-minded  citizens  as  a  mass  continue  to  lack 
confidence  in  works  of  this  type,  the  latter  cannot  be  called  an 
unqualified  success,  no  matter  how  fully  scientific  facts  may  show 
their  adequacy. 

The  Massachusetts  Institute  of  Technology  instituted  the  plan 
of  especially  training  young  men  along  technical  lines,  so  that  they 
might  become  competent  to  serve  as  superintendents  for  water  and 
sewage-purification  works.  In  this  pioneer  work  they  are  entitled 
to  great  credit,  and  their  example  is  already  being  followed  by  similar 
institutions  elsewhere.  This  is  an  important  field  of  technical 
education,  as  a  majority  of  such  technically  educated  men  in  the 
future  will  be  connected  with  the  management,  rather  than  with 
the  construction,  of  works  of  this  type. 

In  passing,  it  may  not  be  amiss  to  say  that  the  technical  managers 
of  works  of  the  type  under  consideration  must  have  other  quali- 


Experimental  Methods  in  Water-  and  Sewage-Works    31 

fications  than  those  of  a  scientific  nature.  They  must  be  able  to 
maintain  amicable  relations  with  executive  superiors,  to  manage 
laborers,  to  keep  records  in  a  manner  fairly  comparable  with  the 
high  degree  to  which  the  art  of  bookkeeping  in  large  business  houses 
has  advanced,  to  prepare  reports  containing  essential  features  in 
explicit  but  terse  terms,  and  to  make  plain  to  non-technical  men 
in  both  public  and  private  capacity  the  more  essential  features  of 
their  own  position  and  of  the  data  by  which  their  efforts  show  what 
is  being  accomplished.  This  type  of  specialists  will  naturally  develop 
in  efficiency  as  their  responsibihties  increase;  but  there  is  still  much 
work  for  the  technical  schools  to  do  in  preparing  young  men  more 
adequately  for  these  duties. 

Tentative  installations. — As  distinguished  from  the  testing  stations 
built  solely  for  the  purpose  of  tests,  there  is,  of  course,  one  other 
method  of  a  somewhat  experimental  nature  by  which  local  data 
are  used  in  determining  whether  large  works  are  most  advanta- 
geously constructed.  I  refer  to  the  plan  of  constructing  works 
gradually,  or  tentatively,  and  of  using  data  from  the  operation 
of  the  first  portion  of  the  installation  to  serve  as  a  guide  in 
arranging  the  details  of  the  portions  subsequently  to  be  built,  and 
also  in  deciding  upon  the  magnitude  of  the  works  sufficient  for  a 
given  capacity  or  to  serve  for  a  given  term  of  years.  This  is  the 
style  of  works,  from  the  experimental  point  of  view,  which  frequently 
obtains  in  Europe,  and  which  will  obtain  in  some  places  in  this 
country.  As  yet  there  has  not  been  a  wide  apphcation  in  America 
of  such  data  obtained  on  a  large  practical  scale,  although,  of  course, 
they  are  availed  of  more  or  less  in  all  works  where  extensions  are 
required.  This  condition  has  been  reached  at  several  sewage- 
works  in  New  England,  and  the  results  of  experiences  in  the  field 
have  been  summarized  by  the  Massachusetts  State  Board  of  Health. 
It  is  gratifying  to  state  that  practical  results  are  in  general  con- 
formity with  the  principles  of  water  and  sewage  purification  as 
developed  by  tests  on  a  small  scale. 

experimental   methods   in   EUROPE, 

In  Europe  the  water-purification  problems  do  not  cover  nearly  so 
wide  or  difficult  a  range  of  natural  conditions  as  those  met  in  America. 
Filtration  has  in  recent  years  not  received  as  much  attention  experi- 


32  George  W.  Fuller 

mentally  as  has  been  the  case  with  sewage-works.  In  earher  years 
however,  experimental  methods  had  much  to  do  with  the  develop- 
ment of  water  filters  abroad.  It  is  not  to  be  forgotten,  furthermore, 
that  in  Germany  much  good  work  during  the  past  dozen  years  has 
been  done  in  developing  the  most  practical  methods  for  removing 
iron  from  ground  waters.  At  present  the  most  interesting  feature 
of  water-purification  developments  in  Europe  refers  to  the  prelimi- 
nary treatment  for  some  of  the  river  waters  which  are  fairly  turbid 
during  freshets,  and  to  efforts  to  sterilize  water  economically  by 
ozone.  The  most  notable  instance  of  the  former  is  at  Suresnes, 
near  Paris,  where  the  Seine  water  below  the  metropolis  is  subjected 
to  filtration  six  times,  the  first  filters  being  of  coarse  gravel  to  effect 
clarification. 

In  England,  which  is  the  home  of  modern  sanitary  engineering, 
sewage-purification  works  have  received  more  attention  than  in 
any  other  country.  The  density  of  population  in  England  and 
the  relatively  small  size  of  its  rivers  have,  of  course,  forced  this  con- 
dition at  an  earlier  date  than  is  generally  true  of  other  countries. 
While  for  some  years  the  English  have  not  contributed  much  on 
the  subject  of  water  filtration,  their  experience  in  the  field  of  sewage 
purification  far  exceeds  that  of  any  other  country.  Experimental 
methods  in  one  form  or  another  have  played  an  important  part 
for  half  a  century,  beginning  with  efforts  to  utihze  the  manurial 
value  of  sewage.  This  is  largely  owing  to  the  differences  in  various 
local  conditions,  especially  topography,  geology,  and  the  compo- 
sition of  the  sewage  as  influenced  by  trade  wastes.  Not  only  have 
the  English  conducted  test  filters  and  other  processes  of  purification 
on  a  small  scale,  but  they  have  also  gathered  many  data  of  great 
value  by  the  operation  of  their  works  in  practice  along  Hnes  which 
enable  current  experiences  to  be  utilized  in  developing  future  works. 

These  data  have  been  so  universally  obtained  in  conjunction 
with  the  operation  of  existing  works  in  practice  that  it  is  very  difficult 
to  ascertain  even  roughly  what  their  cost  has  been.  The  staff  regu- 
larly engaged  in  operating  the  main  works  has  secured  the  technical 
data,  so  that  the  expense  has  been  confined  to  building  the  test 
devices,  relatively  small  in  size,  and  to  a  Httle  extra  labor  for  opera- 
tion.    The  large  mass  of  valuable  testimony  published  in  numerous 


Experimental  Methods  in  Water-  and  Sewage-Works    33 

municij)al  reports  and  by  the  Royal  Commission  on  Sewage  Dis- 
posal shows  what  a  fund  of  knowledge  has  been  accumulated  at 
London,  Salford,  Sutton,  Exeter,  Burnley,  Accrington,  Hudders- 
field,  Leicester,  Birmingham,  Bradford,  Devizes,  Hanley,  and  other 
cities,  and  which  for  most  places  has  been  obtained  with  almost 
no  special  fund  devoted  to  testing  purposes,  comparatively  speaking. 

At  Leeds  the  unusually  thorough  sewage  tests  made  during  the 
past  eight  years  received  appropriations  of  about  $150,000,  some 
two-thirds  of  which  has  been  actually  devoted  to  that  purpose. 
Manchester  has  also  expended  quite  large  sums  for  experimental 
purposes,  although,  for  the  reasons  above  stated,  the  expenditures 
were  by  no  means  commensurate  with  the  information  obtained. 
The  Royal  Commission  on  Sewage  Disposal  in  England  is  under- 
stood to  have  an  appropriation  of  about  $55,000  for  the  expenses 
of  its  own  stafif  and  the  traveling  expenses  of  the  numerous  witnesses 
who  have  appeared  before  it.  There  are  also  special  river  boards 
and  county  councils,  with  excellent  technical  staffs,  which  gather 
many  valuable  data. 

In  France  sewage  purification  has  been  the  subject  of  experi- 
mental study,  beginning  with  the  labors  of  Mille  in  1868  at  Gen- 
nevilliers.  These  tests  resulted  in  the  establishment  of  the  present 
sewage  farms  of  Paris.  Within  the  past  few  years  the  biological 
methods  of  purification  have  received  attention  both  from  the  city 
of  Paris  and  from  the  Department  of  Agriculture.  The  latter  has 
a  general  supervising  control  over  water  and  sewage  matters  outside 
of  Paris,  and  is  devoting  an  appropriation  of  about  $60,000  to  such 
investigations.  Thus  far  these  studies  have  been  made  by  Professor 
Calmette  at  Lille,  as  set  forth  in  his  interesting  progress  report  of 
last  autumn. 

In  Belgium  the  government  is  paying  particular  attention  experi- 
mentally to  the  treatment  of  trade  wastes  at  a  special  station  devoted 
to  that  purpose  at  Verviers. 

The  government  of  Holland  established,  in  1904,  a  sewage-testing 
station  at  Tilburg,  the  cost  of  which  to  date  is  approximately  $15,000. 
No  reports  have  yet  been  pubhshed.  Several  ozone  plants  have 
been  tested  in  Holland,  and  the  city  of  Rotterdam  is  now  arranging 
to  test  a  mechanical  filter  on  the  local  river-water  supply. 


34  George  W.  Fuller 

In  Germany  numerous  experiments  have  been  made  upon  the 
sedimentation  of  sewage  for  purposes  of  clarification,  and  the  so- 
called  biological  methods  have  been  studied  for  some  years,  begin- 
ning in  1895,  when  a  testing  station  was  estabhshed  by  Professor 
Dunbar  at  Hamburg,  which  station  is  still  in  operation.  In  1901 
the  Prussian  government  estabhshed  a  permanent  organization 
for  testing  water-  and  sewage-purification  methods.  This  "insti- 
tute" has  gathered  together  and  pubhshed  the  more  important 
data  as  to  experiences  in  other  countries,  has  conducted  several 
important  sewage-testing  stations  in  the  suburbs  of  Berlin,  and  has 
collated  many  useful  data  as  to  the  sanitary  works  of  the  cities  of 
Prussia  and  neighboring  territory.  This  department  has  an  annual 
appropriation  of  about  $30,000  for  testing,  inspecting,  analytical, 
and  clerical  purposes.  The  sum  devoted  to  testing  purposes  varies, 
but  is  materially  supplemented  by  the  arrangement  of  conducting 
investigations  for  various  local  authorities,  the  expense  for  which 
is  borne  in  part  by  the  community  benefited.  The  department 
also  established  the  custom  of  officially  examining  proprietary 
devices,  largely  at  the  expense  of  the  owners  in  cases  where  the 
devices  seem  to  possess  sufficient  merit.  In  this  way  a  mechanical 
filter  of  the  Jewell  type  was  recently  tested  at  the  Miiggelsee  plant 
of  the  Berlin  water-works.  The  same  filter  is  now  being  tested 
on  the  colored  water  supply  of  Konigsberg. 

The  relative  amounts  of  suspended  matters  deposited  from  sew- 
age at  different  velocities  have  been  studied  carefully  under  vary- 
ing local  conditions  at  Frankfurt,  Cassel,  Hannover,  and  Cologne, 
as  shown  by  the  data  published  in  municipal  reports  and  the  technical 
press.  In  these  cities,  as  in  England,  it  is  difficult  to  ascertain 
the  cost  of  the  tests,  because  so  much  of  the  work  was  done  by  the 
regular  staff  of  the  technical  authorities  of  the  cities.  The  scope 
of  the  tests  has  probably  been  greatest  at  Frankfurt,  including  means 
for  most  easily  removing  sludge,  its  partial  drying  by  centrifugali- 
zation,  and  its  ultimate  disposal  by  incineration  after  mixing  with 
the  city  refuse.  About  $60,000  has  been  spent  at  Frankfurt  on 
these  and  other  sewage  tests,  including  filtration,  within  the  past 
dozen  years  or  more. 

Professor  Dunbar's  activities  in  the  field  of  sewage  purification 


Experimental  Methods  in  Water-  and  Sewage-Works    35 

have  by  no  means  been  confined  to  Hamburg.  His  publications 
show  that  he  has  advised  the  authorities  at  Miihlhausen,  Stuttgart, 
Beuthen,  Unna,  Leipzig,  and  other  places.  In  nearly  every  instance 
he  has  taken  advantage  of  experimental  data  to  ascertain  local 
conditions.  Leipzig  and  Chemnitz  in  Saxony  are  now  conducting 
sewage  tests,  the  appropriations  for  which  are  about  $17,000  in 
each  case,  with  the  engineering  and  analytical  data  secured  by  men 
regularly  employed  by  the  city. 

This  brief  record  of  experimental  methods  as  applied  to  water 
and  sewage  purification  can  hardly  be  brought  to  a  close  without 
reference  to  trade  wastes.  This  feature  in  aggravated  cases  com- 
plicates the  design  of  sewage-works  and  adds  materially  to  the  costs 
of  operation.  Various  industries  require  special  consideration,  as 
shown  by  the  efforts  of  the  river  boards  to  minimize  the  effect  of 
trade  wastes  in  the  streams  of  Lancashire  and  Yorkshire,  England. 
The  removal  of  fats  has  perhaps  received  the  most  attention  along 
this  hne — especially  in  Berlin,  Cassel,  and  Chemnitz  in  Germany, 
Verviers  in  Belgium,  Roubaix  and  Grimonpont  in  France,  and 
Bradford,  Manchester,  and  Oldham  in  England.  Numerous  mill- 
owners  also  recover  grease  from  their  waste  water.  The  extent 
of  some  of  these  investigations  is  indicated  by  the  fact  that  at  Cassel 
a  private  company  is  said  to  have  spent  considerably  more  than 
$100,000  in  unsuccessfully  endeavoring  to  fulfil  a  contract  for  extract- 
ing fats  from  the  city  sewage.  The  only  large  place  where  the 
entire  city  sewage  is  regularly  treated  for  grease  extraction  is  at 
Bradford,  England. 


THE  FUTILITY  OF  A  SANITARY  WATER  ANALYSIS  AS 
A  TEST  OF  POTABILITY.* 

Marshall  O.  Leighton. 

Whosoever  expresses  doubts  concerning  generally  accepted 
ideas  must  be  prepared  to  see  his  statements  misinterpreted  and 
their  application  carried  far  beyond  the  point  at  which  they  were 
aimed,  even  to  the  absurd  and  grotesque.  He  must  not  expect 
that  his  observations  and  deductions  will  be  confined  to  the  Hmits 
prescribed,  even  though  he  resorts  to  every  safeguard  that  his  mother- 
tongue  affords.  More  attention  is  paid  to  the  devious  paths  along 
which  his  statements  may  lead  by  implication  than  to  the  single 
trail  that  he  has  defined  by  precise  guide-posts.  Finally,  such  a 
person  must  sustain  confrontation  by  that  splendid,  indispensable, 
and  all-saving  power  known  as  conservatism.  Therefore  the  author 
of  this  paper  hoists  a  flag  of  truce  while  he  makes  his  preHminary 
declaration,  in  the  hope  that  the  highest  possible  proportion  of  those 
interested  may  not  mistake  his  line  of  march. 

1.  All  contentions  concerning  the  futility  of  sanitary  analyses 
are  applied  strictly  to  waters.  Sewages,  fresh  and  stale,  and  sewage 
effluents  are  expressly  ehminated  from  consideration,  except  in  certain 
cases  where  they  will  be  taken  to  illustrate  the  fact  that  they  may 
occasionally  be  accepted  as  unpolluted  water,  according  to  standard 
methods  of  interpretation. 

2.  //  is  not  contended  that  all  sanitary  water  analyses  are  futile 
irrespective  of  the  conditions  under  which  they  are  made  and  inter- 
preted. In  consistent  studies  of  nitrogen,  as  such,  and  the  changes 
which  take  place  in  its  form,  such  analyses  are  important. 

3.  //  is  admitted  that  in  certain  limited  areas  of  the  United  States 
sanitary  water  analyses  afford  information  by  which  animal  pollu- 
tion may  occasionally  be  detected. 

4.  The  facts  comprised  in  the  foregoing  admission  have  been  a 
stumbling-block  to  chemists  working  with  waters  outside  of  those 
limited  areas. 

*  Received  for  publication,  March  30,  1906. 

36 


Futility  of  a  Sanitary  Water  Analysis  37 

5.  //   is  contended  that  the  sanitary  analysis   offers  nothing  by 
which  one  may  positively  distinguish  between  a  dangerous  and  a  whole-^ 
some  water. 

6.  The  composition  ratios  that  many  good  men  cherish  may  be 
applied  indiscriminately  to  wholesome  waters  and  dilute  sewages. 

7.  The  conventional  method  of  seeking  for  evidences  of  pollution 
by  sanitary  analyses,  or  of  accepting  or  rejecting  a  water  upon  such 
evidence,  is  in  its  broad  and  essential  features  quite  misleading,  too 
frequently  dishonest,  and  in  some  cases  absurd. 

8.  The  dangerous  pollution  of  surface  waters  can  be  discovered 
more  readily,  and  at  far  less  risk  and  expense,  than  by  sanitary  an- 
alysis. 

9.  The  term  "sanitary  analysis"  as  used  in  this  discussion  does 
not  include  tests  for  specific  organisms. 

Standards  of  interpretation  by  which  a  water  may  be  designated 
as  "good"  are  faithfully  met  by  many  waters  undeniably  bad;  con- 
versely the  characteristics  of  a  water  interpreted  as  "bad"  are  pre- 
sented by  many  the  wholesomeness  of  which  cannot  be  questioned. 

There  is  in  the  presidential  address  of  Professor  Leonard  P. 
Kinnicutt,  delivered  before  Section  C  of  the  American  Association 
for  the  Advancement  of  Science,  at  New  Orleans,  La.,  in  December, 
1905,  a  concrete  statement  of  intrepretation  standards.  This  state- 
ment will  be  used  as  a  basis  for  the  comparisons  which  follow  in  this 
discussion.  Such  a  selection  is  made,  decidedly  not  for  the  purpose 
of  controverting  the  statements  or  discrediting  the  position  of  this 
distinguished  authority,  but  rather  because  it  is  the  most  admirable 
resume  that  has  recently  emanated  from  a  highly  respected  and 
competent  source.     The  following  statements  are  quoted: 

(A)  In  fresh  sewage  the  amount  of  nitrogen  as  free  ammonia  is  from  three  to  four 
times  that  of  the  nitrogen  in  the  albuminoid  ammonia,  and  in  sewage  efHuents  from 
20  to  30  times,  while  in  peaty  water,  or  water  containing  an  infusion  of  leaves' 
the  nitrogen  in  the  albuminoid  ammonia  is  from  lo  to  20  times  the  nitrogen  in 
free  ammonia.  Hence,  when  a  surface  water,  not  including  rain  or  snow  water, 
gives  a  greater  amount  of  nitrogen  as  free  ammonia  than  it  does  as  albuminoid  am- 
monia, the  indications  arc  that  the  water  has  certainly  been  polluted  by  sewage,  and 
that  the  source  of  the  organic  matter  is  of  animal  origin.  With  a  large  amount  o^ 
nitrogen  as  albuminoid  ammonia  (over  0.25  milhgram  per  liter)  a  ratio  of  nitrogen 
of  the  free  ammonia  to  the  nitrogen  of  the  albuminoid  ammonia  of  less  than  i  to  5 
is  suspicious. 


//// 


3^ 


Marshall  O.  Leighton 


(B)  Consequently,  while  a  low  ratio  as  i  to  5  between  the  nitrogen  of  the  free 
ammonia  and  the  nitrogen  of  the  albuminoid  ammonia  indicates  pollution,  the  reverse 
cannot  be  said  to  be  a  strong  indication  that  the  water  is  a  normal  water. 

(C)  A  colorless  water  containing  that  amount  of  nitrogenous  matter  represented 
by  0.25  milligram  of  nitrogen  as  albuminoid  ammonia  per  liter  is  looked  upon  with 
suspicion. 

(D)  Free  ammonia  always  indicates  organic  matter  in  the  process  of  decompo- 
sition. In  unpolluted  surface  waters  it  is  rarely  high,  being  removed  almost  aS 
fast  as  formed  by  vegetable  and  animal  organisms  in  the  water,  and  an  amount  of 
nitrogen  as  free  ammonia  above  o .  05  milligram  per  liter  is  unusual,  and,  if  it  does 
occur,  the  water  cannot  be  considered  as  an  unpolluted  water  unless  that  fact  is  clearly 
established  by  other  data. 

Attention  is  then  called  to  seasonal  variations  and  the  increase 
in  free  ammonia  during  the  autumn  in  northern  countries. 

(E)  Concerning  nitrogen  as  nitrites: 

More  than  0.002  milligram  per  Hter  is  an  unfavorable  indication. 

(F)  Concerning  nitrogen  as  nitrates: 

It  is  never  present  in  any  large  amount,  seldom  exceeding  o .  i  milligram  per  liter. 
Higher  amounts  than  this,  being  unusual,  must  be  looked  upon  with  suspicion. 

Professor  Kinnicutt  then  explains  that  the  above  interpretations 
refer  to  reservoir,  pond,  and  lake  waters,  but  that  in  river  waters 

high  nitrogen  as  free  ammonia,  as  albuminoid  ammonia,  and  as  nitrites  charac- 
teristic of  recent  pollution  in  ponds  and  reservoirs  may  be  due  to  the  decomposition  of 
algae  life,  which  was  stimulated  by  the  entrance  of  sewage  in  the  upper  stretches  of 
the  river. 

Accepting  the  above  as  an  authoritative  basis  of  interpretation 
— and  it  is  the  one  which  closely  corresponds  to  that  which  the  writer 
has  found  in  very  general  use — let  us  interpret  a  few  analyses.  Ref- 
erence will  be  made  by  letter  to  the  foregoing  quotations,  so  that 
the  basis  of  each  interpretation  may  be  clearly  defined. 

SERIES  "A." 
Parts  per  Million. 


Date 

Tur- 
bidity 

Color 

Odor 

Nitrogen  as — 

No. 

Albuminoid 
Ammonia 

Free 
Ammonia 

Nitrites 

Nitrates 

Chlorine 

1 
2 
3 

July  II,  1900 
July  20,  1899 
Sept.  14,  1900 

0 
Cons. 

SI. 

0 

1-5 
2. 1 

0 
im 
im 

0.026 

0.330 
0. 114 

0.028 
0.  246 
0. 164 

0 
0 
0 

0 
0 
0 

1.2 
1 . 2 
I.  2 

There  are  presented  in  the  above  series  three  analyses  of  pond 
waters.     All   are    practically  colorless,  and  Nos.   2   and    3   have  a 


Futility  of  a  Sanitary  Water  Analysis 


39 


slightly  moldy  odor.  (A)  In  Nos.  i  and  3  the  nitrogen  as  free 
ammonia  is  greater  than  that  as  albuminoid  ammonia.  (D)  Nos. 
2  and  3  contain  very  much  greater  amounts  of  free  ammonia  than 
0.05  part  per  million.  (A,  second  part)  No.  3  contains  over 
0.25  part  of  albuminoid  ammonia  per  million,  and  the  free- 
albuminoid  ratio  is  i  to  1.3.  No  nitrites  or  nitrates  appear  in  any 
of  the  samples.  According  to  the  above  standards  of  interpreta- 
tion, all  three  of  these  waters  contain  recent  organic  pollution  of 
a  very  unstable  nature  or,  in  other  words,  sewage  pollution;  the 
nitrification  is  proceeding  very  rapidly,  and  the  assimilation  of  nitrites 
and  nitrates  is  accomplished  as  rapidly  as  they  are  formed,  by  an 
abundance  of  organisms.  In  point  of  fact,  these  are  normal  waters 
from  two  storage  ponds  in  Pennsylvania,  in  the  drainage  areas  of 
which  there  are  no  habitations.  It  would  be  difficult  to  specify  con- 
ditions that  would  more  closely  approach  the  ideal  for  upland  con- 
served supply  than  existed  at  these  two  places  at  the  time  these  sam- 
ples were  taken.  No.  i  is  from  Pine  Run  Reservoir,  and  Nos.  2 
and  3  from  Mill  Creek  Reservoir,  both  in  Luzerne  County,  Penn- 
sylvania. 

SERIES  "B." 
Parts  per  Million. 


No. 

Tur- 
bidity 

Color 

Odor 

Nitrogen  as — 

Chlorine 

Total 
Residue 

Imss  on 

Albuminoid 
Ammonia 

Free 
Ammonia 

Nitrites 

Nitrates 

Ignition 

I 
2 
3 
4 
5 

DLst. 
It 

It 

(( 

(t 

13 

IS 
8 
8 

13 

2a 
la 
2a 
la 
la 

0.120 
0.  204 
0. 106 
0. 142 
0.130 

0.006 
0.016 
0.002 
0.026 
0.012 

0.000 
0.000 
0.000 
0.000 
0.000 

0.170 
0. 100 
0. 160 
0. 160 
0. 160 

0.9 
0.9 
0.9 
0.9 
0.9 

69  S 
72.0 
66.  s 
66.5 
69.0 

It).  SO 
20.00 
16.00 
13  so 
20.00 

Series  "B"  contains  analyses  of  samples  taken  from  a  large  lake 
in  September,  1904.  Each  sample  was  distinctly  turbid,  of  low  color, 
and  revealed  an  aromatic  odor.  (C)  The  nitrogen  as  albuminoid 
ammonia  is  in  every  case  less  than  0.25  miUigram  per  liter.  (D) 
The  nitrogen  as  free  ammonia  is  in  all  cases  far  less  than  0.05  milli- 
gram per  liter.  (A)  The  free-albuminoid  ratio  varies  from  1-5.3 
up  to  1-5.5.  (E)  There  are  no  nitrites.  (F)  The  nitrates  run 
somewhat  higher  than  the  standard  set. 

Several  of  the  above  samples  have  all  the  characteristics  of  an 


40 


Marshall  O.  Leighton 


infusion  of  leaves  (A)  except  color.  There  is  nothing  in  the  analyses 
presented,  except  the  unimportant  excess  of  nitrates,  that  does 
not  surpass  on  the  acceptable  side  the  strictest  of  the  interpretation 
standards  above  quoted.  The  samples  were  collected  almost  simul- 
taneously on  September  22,  1904,  from  Lake  Champlain,  within 
the  inclosed  area  lying  between  the  docks  at  Burlington,  Vt.,  and 
the  harbor  breakwater.  Sample  No.  2  was  taken  about  1,000  feet 
away  from  the  outlet  of  the  main  trunk  sewer  of  the  city.  The  remain- 
der were  taken  at  points  less  than  500  feet  away  fom  said  outlet. 
No.  5  being  collected  about  20  feet  from  the  sewer's  mouth. 
Ten  years  previous  to  the  collection  of  these  samples  the  city  of 
Burlington  was  obliged  to  abandon  a  water  intake  situated  at  a  much 
more  favorable  point  than  those  at  which  any  of  the  above  samples 
were  taken.  The  reason  for  the  change  was  the  high  rate  of  intes- 
tinal disease  morbidity  in  the  city,  which  was  markedly  decreased 
afterw^ard.  What,  then,  shall  we  say  of  sanitary  analysis  as  an 
index  of  pollution  at  Burlington  ?  Bacteriological  examination  reveal- 
ed the  abundant  presence  of  B.  coli  in  all  the  samples  reported  in 
Series  "B,"  and  the  discharge  of  sewage  into  the  lake  was  a  matter 
of  casual  observation.  Therefore  no  one  was  deceived  by  the  nitro- 
gen determinations.  One  may  very  properly  question  whether 
sanitary  analyses  may  not  be,  under  less  fortunate  circumstances, 
an  actual  danger  to  public  health. 
Let  us  now  examine  series  "C." 

SERIES  "C." 
Parts  per  Million. 


No. 

Nitrogen  as — 

CUorine 

Total 
Residue 

Loss  on 
Ignition 

Albuminoid 
Ammonia 

Free 

Ammonia 

Nitrites 

Nitrates 

I 

2 
3 
4 
5 

0.17 
0. 10 
0.07 
0.18 
0.06 

0.01 
0.01 
0. 12 
0.09 
tr. 

0 
0 
0 
0 
0 

1 .00 
1. 00 
I  25 
2.25 
3  00 

2-5 

50 
4.0 

50 
50 

83 

78 

219 

73 
32 

25 

37 
42 
24 
15 

Records  of  color  and  odor  are  unfortunately  absent,  but  from 
independent  sources  comes  the  assurance  that  none  of  the  samples 
were  highly  colored,  and  the  predominating  odor  is  faintly  earthy. 
According  to  the  standards  of  interpretation,  Nos.  i  and  2  were  in  good 


Futility  of  a  Sanitary  Water  Analysis 


41 


condition  at  the  time  of  analysis  (C  and  D).  The  ammonias  arc 
low,  and  (A)  the  free-albuminoid  ratio  is  excellent.  (F)  It  would 
appear  from  the  large  amount  of  nitrates  and  the  high  chlorine 
that  the  water  has  at  some  time  been  polluted,  but  has  become 
well  purified.  Nos.  3  and  4  are  waters  that  have  been  in  bad 
"company"  (F),  and  the  free-albuminoid  ratios,  especially  that  of 
No.  3,  show  recent  pollution  (A).  No.  5  (see  high  nitrates  and  chlo- 
rine) appears  to  be  an  excellent  example  of  a  water  purified  by  run- 
ning in  a  stream-bed  through  a  long  stretch  of  unoccupied  country 
below  some  initial  seat  of  infection.  Note  the  high  nitrates.  The  truth 
is  that  all  these  waters  were  taken  from  unpolluted  streams  in  the 
mountain  districts  of  the  Potomac  drainage  area  in  Virginia  and 
West  Virginia. 


SERIES  "D." 
Parts  per  Million. 


Nitrogen  as — 

u 
■0 

Hardness 

X 

Date 

■B  a 

rt 

0 

13 

"o 
U 

1_ 

0 

II 
1^ 

a 
0 

C/1 

2 

2; 

3 
0 

e2 

>, 

•a 

J3 

3 
2 

Aug.  22,  1903.  . 

1 

4 

0 

0.056 

0.050 

0. 

0.025 

1.73 

68 

46 

8.3 

1 

I 

Sept.  28,  1903. . 

40 

l.S 

0 

0.341 

0.055 

0. 

0. 125 

3.64 

121 

52 

23.0 

7 

2 

Oct.  24.  1903. . 

5 

26 

0 

0. 140 

0.04 

0. 

0. 125 

S.90 

130 

32 

330 

35 

3 

Nov.  17, 1903. . 

60 

."il 

M 

0. 226 

0.084 

O.OOI 

0.144 

S-io 

144 

28 

330 

3 

4 

Dec.    8,  1903. . 

I 

9 

0 

0.054 

0.034 

0. 

0.025 

2.80 

77 

29 

3SO 

1:15 

S 

Analysis  No.  i,  in  Series  "D,"  indicates  a  practically  colorless 
and  odorless  water,  with  nitrogen  in  all  four  forms  low  in  amount. 
The  chlorine,  too,  is  low  and  practically,  the  only  suspicious  feature 
about  the  statement  is  the  free-albuminoid  ratio  (A). 

No.  2  is  a  turbid  water  of  moderate  color.  The  amount  of  nitro- 
gen as  albuminoid  ammonia  is  high,  but  the  free-albuminoid  ratio 
("A,"  last  part)  is  1:7.  (F)  Nitrogen  as  nitrates  is  high.  It  is  not 
a  very  bad  water  according  to  the  interpretation,  yet  there  are  evi- 
dences that  some  swamps  are  tributary  to  the  point  at  which  it  was 
taken. 

No.  3  looks  suspicious  because  of  the  free-albuminoid  ratio  (A), 
the  moderately  high  free  ammonia  (D)  and  nitrates  (F),  and  the 
high  chlorine. 


42 


Marshall  O.  Leighton 


No.  4  is  a  turbid,  highly  colored  water,  with  a  moldy  odor,  a  bad 
free-albuminoid  ratio  (A),  an  appearance  of  nitrites,  and  high  nitrates 
(F)  and  chlorine.      It  is  a  thoroughly  "  suspicious  "-looking  water. 

No.  5  has  practically  the  same  characteristics  as  No.  i. 

The  object  of  introducing  these  tables  is  not  so  much  to  show 
the  misleading  character  of  the  data  as  to  call  attention  to  their 
variations.  Here  are  five  analyses  of  a  normal  water,  taken  at  the 
same  point  from  a  small  stream  draining  an  uninhabited  wilderness. 
Yet  only  two  of  them  possess  a  resemblance  of  uniformity,  and  the 
free-albuminoid  ratios  vary  from  those  of  a  dilute  sewage  to  those 
of  a  potable  water.  The  variations  in  chlorine,  too,  are  interesting, 
and  they  lead  one  to  speculate  upon  the  actual  normal  chlorine 
value  for  this  region.  The  samples  were  taken  from  the  head  waters 
of  Green  River  in  Casey  County,  Kentucky. 


SERIES " E 

M 

Parts  per  Million. 

Nitrogen  as — 

3 

Hardness 

T3 

1 

■a  rt 

C3 

-a 

E 

Is 

Date 

'§'3 
.S  ° 
6  2 

•a 
0 

E 

J 

•a 

« 

3 

S 

0 
■0 

^A 

'u. 

0 
2 

3 
,0 

v.^, 

E 

3 

H 

0 

0 

< 

£ 

z 

2 

u 

h 

< 

l^ 

fa 

Z 

Sept.  2o,  1903 

20 

20 

0 

0.272 

0.130 

0.009 

0.22s 

S.6 

130 

6s 

24 

1:2 

I 

20 

17 

0 

0.302 

0.130 

0.002 

0.187 

5-5 

128 

60 

14 

1:2.3 

2 

Series  "E"  presents  several  points  of  interest.  Both  waters 
are  of  moderate  color  and  turbidity,  and  have  no  odor.  According 
to  the  standards  of  interpretation.  No.  i  is  a  recently  polluted  water. 
It  contains  a  large  amount  of  nitrogen  as  albuminoid  ammonia, 
(A)  and  the  free  albuminoid  ratio  is  i  to  2.  Free  ammonia  is  very 
high  (D).  Nitrites  and  nitrates  (E  and  F)  are  both  high.  On  the 
whole,  the  water  may  be  said  to  be  both  recently  and  remotely  pol- 
luted. No.  2,  although  somewhat  similar,  is  superior  in  some  respects. 
The  free-albuminoid  ratio  is  i  to  2.3.  Free  ammonia  is  the  same, 
while  nitrites,  nitrates,  and  chlorine  are  lower,  though  the  last  is 
not  significantly  different.  The  fact  that  in  No.  2  the  albuminoid 
ammonia  is  higher  than  No.  i  is  responsible  for  the  better  ratio 
in  the  former. 

One  might  readily  infer  that  both  samples  were  taken  from  the 


Futility  of  a  Sanitary  Water  Analysis 


43 


same  stream  at  the  same  place.  "Bad"  water  No.  i  was  taken 
from  Kentucky  River  above  the  city  of  Frankfort  at  the  water-works 
intake.  "Bad"  water  No.  2  is  from  Kentucky  River  below  the 
Frankfort  sewers.  Note  that  the  dates  of  sampHng  are  the  same. 
The  important  feature  of  Series  "E"  is  that  here  is  a  water,  or  a 
dilute  sewage,  taken  below  the  sewers  of  a  city  of  20,000  inhabitants 
showing  practically  the  same,  if  not  a  better  condition,  according 
to  the  interpretation  standards,  than  another  sample  taken  from 
the  stream  above  the  sewers. 

Cases  similar  to  the  above  are  very  common.  Two  good  examples 
are  presented  in  Series  "F." 


SERIES " F " 
Parts  per  Million. 


Date 


Nitrogen  as — 

Hardness 

!2 

3 

.3 

■3 

1 

0 

-0 
■3 -a 

1 

5^ 

0 

E 

a*  G 

1 

•n 

0 

IS 
0 

-a 
1 

•a 

1 
IS 
1= 

e 

•i 

B 

a 


Mississippi  River  at  Brainerd,  Minn. 


Nov.    3,  1904 

((      ((      ii 

Feb.  28,  1905 


14 

112 

2V 

0.382 

0.022 

0 

0.03 

I.O 

ISO 

100 

4 

1:17 

IS 

112 

2V 

0.382 

0.040 

0 

0.03 

I.O 

142 

100 

4 

1:9.5 

10 

40 

IV 

0.256 

0.051 

0.002 

0.04 

1-4 

i8S 

161 

i:S 

10 

40 

iv  +  iM 

0.300 

0.040 

tr. 

0.04 

1.8 

188 

162 

1:75 

Above  town 
Below     •' 
Above     " 
Below     " 


St.  Louis  River  at  Cloquet,  Minn. 


Oct.  31,  1904 
Feb.  25,  1905 


17 

292 

2V 

0.502 

0.036 

0 

0.04 

1.6 

121 

34 

I 

1:14 

19 

112 

2V 

0.302 

0.032 

tr. 

0.08 

1.8 

1S6 

98 

1:9.4 

-7 

112 

2V 

0.322 

0.152 

tr. 

0.08 

1 .2 

I  SI 

104 

4 

1:2 

-7 

112 

2V 

0.302 

0.032 

tr. 

0.08 

1.8 

156 

98 

7 

i:  10 

Above  town 
Below      ' 
Above      ' 
Below 


SERIES  "G." 
Parts  per  Million. 


>• 

■| 
13 

u 

3 

3 

Nitrogen  as — 

.3 

u 

0 
u 

3 

e2 

1 

B 

Is 

E 

3 

0 

■0.3 

5 

0 

B 
-J 

2 

8 

Date 

1.  . 

2.  . 

3-- 

4.. 
$■■ 

6.. 

7.- 

SI. 
Sl. 
SI. 
SI. 

si. 

SL 

0 
0 
0 

0. 1 
0 

0. 1 
0. 2 

0.480 
0.37s 
0.656 

0395 
0.53s 
I  .  200 
0.  511 

0.080 
0.013 
0. 104 
0.026 
0.026 
0033 
0.085 

0 
0 
0 
0 
0 
0 
0 

0.461 

0.37s 

0.307 
0. 142 
0.472 
0.349 

70 
76 

59 
74 
67 

S7 
68 

634 
612 
818 
790 
566 
850 
604 

1:6 

1:29 

1:6 

i:iS 
1:21 
1:36 
1:5 

Mar.  s.  1808 
"     II,  1898 

Apr.     9,  1898 

Mar.  II,  1898 
"  18.  I«9» 
"      25.  1898 

Apr.      I,  1898 

44  Marshall  O.  Leighton 

The  waters  represented  in  Series  "G"  are  typical  of  the  western 
prairie  states.  In  such  waters  large  amounts  of  organic  matter  are 
always  present  even  in  the  absolutely  uninhabited  regions.  The  sam- 
ples above  reported  are  taken  from  a  stream  that  drains  a  large  area 
underlaid  with  saline  deposits,  which  account  for  the  high  chlorine 
content.  Such  waters  absolutely  controvert  paragraph  (C)  of  the 
foregoing  interpretations.  It  will  be  noted  that  the  free-albuminoid 
ratio  specified  in  "A"  (last  part)  is  satisfied  by  all  of  the  analyses. 
There  are  no  nitrites,  and  only  in  Nos.  i,  3,  and  7  does  the  amount 
of  nitrogen  as  free  ammonia  exceed  the  standard  set  in  "  D."  Nitro- 
gen as  albuminoid  ammonia  and  nitrates  are  not  high  for  prairie 
waters.  Although  some  of  the  analyses  look  "good,"  the  samples 
were  all  grossly  polluted.  The  first  three  samples  were  taken  from 
Kaw  River  i .  5  miles  above  Lawrence,  Kans.,  and  contain  the 
residue  of  pollution  from  the  sewers  of  Topeka.  The  last  four 
samples  ware  taken  from  the  same  stream,  but  the  point  was  300 
feet  below  the  outlet  sewer  of  Lawrence.  It  will  be  seen  that  the 
samples  from  below  town  present  a  better  analytical  appearance 
than  those  from  above. 

We  will  now  consider  ground  waters.  In  the  address  above 
quoted  there  are  the  following  statements: 

(A)  ....  It  may  be  said  that  the  best  ground  waters  should  certainly  con- 
tain not  over  o.  oi  milligram  of  nitrogen  as  free  ammonia,  or  (B)  over  o .  02  milligram  of 
nitrogen  as  albuminoid  ammonia,  (C)  no  nitrogen  as  nitrites,  (D)  not  over  o .  01  milligram 
of  nitrogen  as  nitrates  in  a  liter  of  water,  and  (F)  chlorine  not  above  the  normal  of 
the  region.  When  a  water  contains  (F)  more  than  0.05  milligram  of  nitrogen  as 
free  ammonia,  and  (G)  o . 08  milligram  of  nitrogen  as  albuminoid  ammonia,  or  0.12 
milligram  of  nitrogen  as  albuminoid  ammonia,  even  if  the  free  ammonia  occurs  in 
very  small  amounts,  it  is  a  sign  of  imperfect  filtration  or  of  subsequent  pollution,  and 
consequently  such  water  should  not  be  used  for  household  purposes. 

It  is  more  difficult  to  determine  the  presence  of  pollution  in  a 
ground  water  by  inspection  than  in  a  surface  water,  and  in  dis- 
cussing ground-water  analyses  one  is  sometimes  unable  to  make 
a  definite  statement  concerning  direct  pollution.  We  can,  for  exam- 
ple, in  the  case  of  a  surface  water  state  that  if  the  water  is  from 
an  uninhabited  region  it  must  be  unpolluted  with  animal  waste. 
On  the  other  hand,  there  is  but  one  certain  method  of  determining 
the  healthfulness  of  a  ground  water.     This  method  has  been  accepted 


Futility  of  a  Sanitary  Water  Analysis 


45 


by  chemists  and  sanitarians  the  world  over  as  being  the  best  test 
of  the  wholesomcncss  of  all  waters,  whether  from  the  surface  or 
from  the  ground — namely,  the  incidence  of  typhoid  fever  and  other 
water-borne  diseases  among  the  habitual  users  of  such  water  as  a 
beverage.  Although  there  are  on  record  many  hundred  analyses  of 
ground  waters,  which  would,  by  the  interpretation  standards  above  set 
forth,  be  classified  as  polluted,  but  which,  judging  from  the  location 
and  all  the  surroundings,  might  be  regarded  as  wholesome,  never- 
theless the  basis  of  the  statements  made  in  the  following  paragraphs 
will  rest  solely  upon  the  typhoid  rate  prevailing  among  the  users  of 
the  various  waters.  The  first  series  of  ground-water  analyses  to  be 
discussed  are  grouped  in  the  following  table: 

SERIES  "H." 
Parts  per  Million. 


Nitrogen  as — 

Chlorine 

Total 
Residue 

Date 

Albuminoid 
Ammonia 

Free 
Ammonia 

Nitrites 

Nitrates 

Aug.  30,  1905. .  . . 

Oct.  10,  1900 

May  18,  1905 

"      18,     "     .... 

May  II,  1898 

Dec.  8,  1904 

0.044 
0.082 
0.048 
0.216 
0.058 
0. 192 

0.136 
0.054 
0.032 
0.032 
0.062 
0.032 

0.008 
0.006 
0.009 
0.009 
0.007 
0 

0.152 
9  394 
9.000 
4.  200 
0.600 
0.080 

1.80 

6.40 

12.00 

63.25 

10.00 

7.50 

341  2 
324. 8 
427.2 
1042.4 
432.8 
3S30 

It  will  be  seen  from  the  above  that  all  the  waters  analyzed  con- 
tained more  nitrogen  in  all  the  specified  forms  than  would  be  allow- 
able under  the  standards  of  interpretation  above  quoted.  The 
analyses  presented  represent  either  the  city  supply  of  Rockford, 
III.,  or  that  from  private  wells  which  are  largely  used  in  that  place. 
They  are  in  all  cases  ground  waters,  and  are  similar  in  character 
to  waters  from  various  wells  in  that  region.  The  writer  has  before 
him  135  analyses  of  well  waters  from  Rockford,  by  far  the  majority 
of  which  present  characteristics  similar  to  those  presented  in  Series 
"H."  Rockford  has  the  lowest  typhoid  fever  death-rate  of  any 
city  in  the  United  States  having  a  population  of  30,000  or  over. 
It  will  be  noted  in  the  various  pubhshed  tabular  statements,  such 
as  that  presented  by  Mr.  George  C.  Whij)ple  in  the  report  of  the 
Commission  on  Additional  Water  Supply  for  the  City  of  New  York 
that  Rockford  almost  invariably  stands  at  the  foot  of  the  list,  with 
a  death-rate  of  about  6  per  100,000. 


46 


Marshall  O.  Leighton 


Let  us  consider  another  case: 


SERIES  "I." 
Parts  per  Million. 


Organic 
Nitrogen 

Nitrogen  as — 

Chlorine 

Total 
Residue 

Oxygen 

Consumed 

Albuminoid 
Ammonia 

Free 
Ammonia 

Nitrites 

Nitrates 

2.75 
2.86 
2.68 

0.280 

I.  TOO 

0.610 

0.07 
1. 100 
0. 142 

0.001 

O.OOI 
O.OOI 

0.57 
0.24 

0.37 

7.5 
12.0 

6.3 

368 
569 
400 

3-73 
301 
2.6s 

The  analyses  in  the  above  table  represent  the  city  water  of  Des 
Moines,  Iowa.  The  first  represents  the  water  from  the  large  well; 
the  second,  from  the  small  well;  while  the  third  is  an  average  of 
42  analyses  of  the  supply,  all  made  in  the  year  1897.  Here  again 
comment  upon  the  divergence  of  these  figures  with  those  given  in 
the  standards  of  interpretation  is  unnecessary.  Des  Moines,  Iowa, 
is  one  of  the  most  fortunate  cities  in  the  country  from  the  stand- 
point of  typhoid  rates. 

Series  "  J  "  contains  respectively  the  average  of  analyses  made  from 
the  Oconee  and  the  Shetucket  wells  of  the  Brooklyn  water  supply. 
Throughout  the  entire  period  between  1897  and  1902  it  will  be 
noted  that  in  neither  case  does  the  average  number  of  bacteria  exceed 
50  per  c.c,  and  there  were  no  positive  tests  for  coli  during  the  entire 
period  of  investigation.  Nevertheless,  the  nitrogen  determinations^ 
according  to  the  above  standards  of  interpretation,  would  condemn 
this  water. 

SERIES  "J." 
Parts  per  Million. 


Nitrogen  as — 

Hardness 

u 

4j  *.» 

•0.2 

cfl 

3 
•a 

m 

K 

l"*^ 

>, 

■33 

"a 

C/3 

>t 

u 

Q 

'■3 

si 

E 

£ 

ffi 

a 

'c 

.2 

J2 

5 

"o 

-1 

b 

13 

0 

"3 

ES3 

a 
0 

u 

er  c 
test 
in  c 

H 

0 

0 

^ 

£ 

2 

K 

U 

H 

^ 

:< 

(— 1 

m 

Oh 

Oconee  WeUs  .  .  . 

I 

6 

0 

0.20 

2.65 

O.OOI 

O.OI 

4.8 

148.7 

lOI  .  0 

50 

0.57 

33 

0 

Shetucket  Wells  . 

10 

25 

0 

o.ois 

0402 

0.012 

O.OI 

264.2 

713-5 

80.6 

1594 

2.02 

50 

0 

Another  example  is  contained  in  Series  "J."  The  samples  were 
taken  from  an  isolated  well  at  St.  Cloud,  Minn.,  and  it  must  be  con- 
fessed that  the  analyses  have  an  unfavorable  appearance,  especially 


Futility  of  a  Sanitary  Water  Analysis 


47 


by  reason  of  the  amounts  of  nitrites.  The  water,  however,  is  abso- 
lutely unpolluted.  The  first  sample  contained  two  bacteria,  and  the 
second,  one  bacterium  per  c.c.  Only  one  species  was  represented, 
which  was  not  B.  coli. 


SERIES  "T." 
Parts  per  Million. 


Date 

>. 

Nitrogen  as — 

3 

Hardness 

■0.2 

C   0 

.2 

'5 
0 

>. 

"2 
IS 
3 

7! 

0 

•a 

11 

5 

E 

''J 

10 

1 

_o 

0 

11 

H 

U 

U 

Uh 

'i^ 

z 

U 

H 

< 

2; 

Sept.  4, 

1904 

0 

0 

0 

0.042 

0.014 

0.015 

7.00 

3-0 

256 

156 

17 

"     4. 

*' 

0 

0 

0 

0.036 

0.014 

0.066 

7.00 

30 

276 

IS7 

16 

For  a  final  example  the  following  analysis  is  submitted,  the  expres- 
sion of  results  being  in  terms  of  parts  per  million : 

Free  ammonia 0.012 

Albuminoid  ammonia 0.012 

Nitrites    .      .  0.00 

Nitrates "strong" 

The  above  water  is  from  a  well  in  Rochester,  Minn.  It  will 
be  seen  that  the  ammonias  are  extremely  low  in  amount,  and  the 
nitrites  and  nitrates  practically  absent.  It  is  a  water  which  con- 
forms in  all  respects  to  the  standards  above  quoted,  yet  it  contained 
1,570  bacteria  per  c.c,  and  an  abundance  of  B.  coli. 

There  remain  for  consideration  the  artesian  or  deep-seated  rock 
waters.  Examples  might  be  cited  in  the  support  of  the  general 
contention  of  this  paper,  but  it  will  be  distinctly  preferable  merely 
to  refer  to  a  paragraph  in  the  address  above  quoted,  as  follows: 

Unfortunately,  however,  in  the  study  of  artesian  water  perplexing  chemicaj 
and  bacteriological  results  are  often  obtained.  In  artesian  waters  so  situated  that 
surface  pollution  seems  impossible,  amounts  of  nitrogen  as  free  ammonia,  as  nitrites, 
and  as  nitrates  have  often  been  found  which,  if  occurring  in  ground  waters,  would 
cause  them  to  be  considered  as  polluted.  The  nitrogen  of  the  nitrates  in  these  waters 
may  be  due  to  fossil  remains,  and  the  nitrogen  as  nitrites  and  as  free  ammonia  to  the 
reduction  of  the  nitrates  by  chemical  action,  as  contact  with  iron  sulphide,  and  the 
occurrence  of  the  nitrogen  as  free  ammonia  also  sometimes  to  some  salt  of  ammonia 
existing  in  the  strata  through  which  the  ground  water  passes.  On  this  account  the 
determination  of  the  nitrogen  content  does  not  give  as  satisfactor}'  data  from  which 
to  draw  conclusions  as  those  obtained  from  the  analysis  of  ground  water. 


48  Marshall  O.  Leighton 

The  contentions  of  Professor  Kinnicutt,  set  forth  in  the  above 
paragraph,  are  supported  by  abundant  evidence,  and  it  constitutes 
as  strong  a  statement  in  support  of  the  vv^riter's  position  as  he  himself 
could  ever  hope  to  draw ;  therefore  the  paragraph  is  submitted  without 
discussion  or  amendment. 

The  analyses  quoted  in  the  following  paragraphs  are  merely 
the  chosen  representatives  of  a  great  number  that  give  the  same 
testimony.  They  all  show  clearly  the  amount  and  condition  of 
the  nitrogenous  matter,  and  can  be  used'  to  dififerentiate  in  some 
small  degree  between  a  comparatively  stable  and  an  unstable  form 
of  organic  matter  in  water.  But  they  show  further  that  all  those 
finely  drawn  distinctions  by  which  we  are  supposed  to  determine 
whether  or  not  such  organic  matter  is  of  benign  or  dangerous  origin 
are  too  precarious  to  be  seriously  considered.  In  every  case  it  is 
easy  to  find  a  host  of  discrediting  exceptions ;  and  when  we  go  beyond 
the  confines  of  New  England  and  the  country  immediately  there- 
about, and  especially  when  we  select  our  samples  from  the  South 
or  the  Middle  West  or  Far  West,  those  exceptions  become  the  rule. 

That  real  man,  the  lamented  friend  of  the  most  of  those  con- 
tributing to  this  volume,  Dr.  Thomas  M.  Drown,  found  not  a  few 
places  in  or  near  New  England  where  his  standards  of  interpreta- 
tion were  useless.  For  example,  many  of  us  remember  hearing 
him  say  that  the  polluted  water  of  the  Hudson  above  Poughkeepsie, 
N.  Y.,  does  not  show  upon  sanitary  analysis  any  traces  of  sewage 
matter.  Yet  neither  he  nor,  it  is  believed,  the  most  enthusiastic  sup- 
porter of  nitrogen  determinations  would  accept  that  raw  water  as  a 
beverage.  In  later  years,  not  many  months  before  Dr.  Drown's 
death,  the  writer  discussed  with  him  the  advisability  of  making  an 
extended  series  of  sanitary  analyses  upon  the  waters  of  the  Lehigh 
River  basin.  Dr.  Drown  approved  of  a  sanitary  survey,  but  failed  to 
see  any  promise  in  the  analytical  work.  He  ended  his  discussion  of 
the  matter  by  saying:  "My  long  experience  in  this  hne  of  work  has 
impressed  me  with  many  doubts  concerning  its  value." 

The  practice  of  making  sanitary  analyses  and  of  judging  the 
potability  of  a  water  from  them  has  cost  many  lives.  The  cases  are 
numerous  and  too  well  known  to  require  discussion.  In  really 
competent    hands    such    analyses    do   not    usually    produce   serious 


Futility  of  a  Sanitary  Water  Analysis  49 

results,  because  they  are  relegated  into  their  proper  place;  but  the 
supposedly  competent  hands  are  frequently  brought  to  book.  Let 
us  review  an  instance. 

In  the  memorable  case  of  the  State  of  Missouri  vs.  the  State 
of  Illinois  and  the  Sanitary  District  of  Chicago  there  was  introduced 
into  evidence  the  testimony  of  a  professor  of  chemistry  who  qualified 
as  an  expert  by  relating  all  sorts  of  educational  experience,  both 
foreign  and  domestic.     Cross-examination  developed  the  following: 

Q.:  Taken  in  the  abstract,  without  reference  to  anything  else  than  the  elements 
of  pollution  stated  by  you,  to  wit,  free  ammonia,  0.063,  nitrites  0.002,  albuminoid 
ammonia  0.552,  nitrates  0.39,  are  those  figures  sufficient  to  warrant  you  in  a  con- 
clusion as  to  the  potability  of  the  water  ? 

A.:  I  think  so. 

Q. :  What  is  your  conclusion  ? 

A.:  It  is  a  potable  water 

Q.:  Do  you  consider  a  water  having  the  following  constituents  potable,  namely, 
free  ammonia  0.217,  nitrites  0.013,  albuminoid  ammonia  0.676,  nitrates  0.6? 

A.:  No,  sir. 

Q. :  On  what  account  ? 

A.:  Because  the  free  ammonia  has  gone  beyond  0.2,  and  the  nitrites  are  up  in 
the  second  place,  whereas  potable  water  should  not  have  free  ammonia  very  much  above 
o.  i;  and,  in  fact,  if  the  nitrites  are  measurable  at  all,  we  usually  condemn  the  water; 


Here  is  a  man  who  gave  two  positive  opinions  concerning  pota- 
bility of  two  waters,  from  the  bare  statement  of  the  four  nitrogen 
determinations.  He  did  not  think  it  necessary  to  take  into  account 
the  other  conventional  statements.  There  are  two  lamentable  features 
of  this:  first,  he  is  teaching  sanitary  chemistry  to  students  in  a  high- 
grade  university;  second,  he  is  only  one  of  a  large  number  of  persons 
similarly  situated  who  are  addicted  to  precisely  the  same  absurdities. 

It  is  anticipated  that  one  of  the  principal  objections  made  to  the 
foregoing  discussion  will  be  that  the  examples  given  are  exceptional 
cases,  and  that  a  far  greater  number  of  examples  can  be  adduced 
which  will  support  the  standards  of  interpretation;  that  a  few  excep- 
tions do  not,  in  science,  destroy  a  theory,  and  that  a  great  mass  of 
data  collected  during  past  years  should  be  accepted  as  the  deter- 
minative basis.  The  cases  presented  for  illustration  are  not  excep- 
tional ones,  nor,  indeed,  are  they  the  best  that  might  have  been  selec- 
ted for  the  purposes  of  this  paper.     If,  however,  we  admit,  for  the 


V 


5©  Marshall  O.  Leighton 

purposes  of  argument,  that  they  may  be  exceptional,  the  contentions 
of  the  writer  are  not  damaged  thereby.  It  should  be  remembered 
that  in  making  sanitary  analyses  we  are  not  developing  a  scientific 
theory  that  must  stand  or  fall  according  to  the  weight  of  cumulative 
evidence  for  or  against,  but  we  are  trying  to  determine  whether 
or  not  the  use  of  that  water  will  cause  sickness  and  death.  This  is 
a  positive  purpose ;  and  if  it  is  admitted  that  there  can  be  exceptions, 
even  though  they  be  few,  the  whole  scheme  of  analytical  procedure 
fails  of  that  purpose.  Exceptions  are  not'  predestined,  and  in  this 
case  cannot  be  guided  or  defined.  The  chemist  who  calls  a  water 
''good"  upon  the  evidence  presented  by  his  nitrogen  determinations 
has  no  means  of  knowing  whether  or  not  this  water  may  be  one  of 
the  exceptions.  Supposing  it  be  polluted  like  the  Lake  Champlain 
samples  in  Series  "B,"  and  a  family  or  a  community  accepts  the 
favorable  opinion  of  the  chemist  and  is  stricken  with  typhoid  fever 
— think  you  that  that  chemist  will  be  justified  by  appearing  before 
those  bereaved  relatives  and  reciting  the  fact  that  the  great  mass 
of  evidence  sustains  certain  bases  of  interpretation,  and  the  scat- 
tering exceptions  do  not,  from  a  scientific  standpoint,  destroy  the 
integrity  of  the  theory  ?  The  most  befitting  remark  at  this  junc- 
ture seems  to  be  the  old  proverb:  "A  chain  is  no  stronger  than  its 
weakest  link." 

After  all,  perhaps  the  strongest  indictment  of  the  sanitary  analy- 
sis is  that  it  is  unnecessary  for  the  purposes  for  which  it  is  generally 
used.  No  one  will  question  its  value  in  sewage  experiments,  but 
when  the  purpose  is  to  determine  whether  or  not  a  water  be  potable, 
there  are  more  satisfactory  ways  of  solving  the  problem  than  by 
making  the  conventional  grind  of  nitrogen  determinations,  even 
though  it  be  admitted  for  the  moment  that  those  determinations 
fulfil  all  the  great  purposes  claimed  for  them. 

It  may  be  accepted  as  axiomatic  that  no  river,  upon  the  drainage 
area  of  which  there  is  any  population,  will  furnish  a  water  fit  for  domes- 
tic consumption  in  its  raw  state.  That  this  shall  hold  true  it  is  not 
necessary  that  the  population  shall  be  gathered  into  cities  and  be 
provided  with  sewerage  systems.  Rural  population,  even  though 
widely  scattered,  is  dangerous.  Wherever  people  live  along  the  banks 
of  a  stream  there  will  always  be  dangerous  pollution.     Indeed,  the 


Futility  of  a  Sanitary  Water  Analysis  51 

natural  drainage  from  occupied  land  is  not  always  innocuous;  but 
when  this  is  combined  with  those  direct  and  generally  surreptitious 
pollutions,  the  effect  is  sometimes  more  acute  than  that  produced  by 
the  everyday  discharges  from  a  city  sewer.  It  will  be  necessary  merely 
to  recall  the  history  of  some  of  our  classic  typhoid  epidemics  to  dem- 
onstrate this.  The  Plymouth,  Pa,,  epidemic  was  caused  by  a  single 
focus  of  infection  upon  a  sparsely  settled  drainage  area.  The  New 
Haven  epidemic  arose  from  a  similar  cause,  and  upon  a  drainage 
area  not  only  sparsely  settled,  but  supposedly  well  protected.  It 
is  especially  significant,  too,  that  the  Lowell  and  Lawrence  epidemics 
did  not  have  as  their  immediate  cause  the  infected  sewage  from  cities 
above  on  the  Merrimack,  but,  as  shown  by  Professor  Sedgwick, 
from  one  or  two  incidental  pollutions  of  Stony  Brook.  The  same 
principles  apply  forcibly  to  the  more  recent  epidemics  at  Butler, 
Pa.,  and  Ithaca,  N.  Y.  These  remarkable  instances  illustrate  the 
dangers  of  surface-water  drainage  from  sparsely  settled  countries; 
it  is  obviously  unnecessary  to  discuss  similar  dangers  from  water 
into  which  city  sewage  is  poured.  The  question  whether  a  river 
water  will  purify  itself  from  such  discharges  in  a  given  distance 
below  a  sewer  outlet  does  not  enter  even  remotely  into  this  considera- 
tion, for,  assuming  that  the  sewage  discharges  would  be  purified, 
the  incidental  pollutions  above  a  water  intake  would  still  constitute 
a  grave  danger.  In  the  case  of  the  Lowell  and  Lawrence  epidemics, 
for  example,  perfect  sewage  purification  at  Manchester,  Concord, 
and  other  cities  on  the  Merrimack  above  the  Lowell  intake  would 
not  have  prevented  the  scourge  of  typhoid.  Therefore  it  is  contended 
that,  if  we  accept  the  principle  that  no  surface-water  draining  from 
an  inhabited  area  is  safe  in  its  raw  state  for  domestic  consumption, 
we  shall  err,  if  we  err  at  all,  upon  the  safe  side,  and  there  is  no 
question  that  we  shall  save  lives.  What,  then,  is  the  necessity  for 
analyzing  river  water  for  pollution,  if  we  are  all  agreed  that  it  must 
inevitably  be  polluted  ? 

Upland  conserved  supplies  present  a  different  phase  of  the  ques- 
tion. If  a  drainage  area  contributing  to  a  reservoir  is  in  primeval 
condition  with  respect  to  population,  it  is  generally  admitted  that  the 
water  must  be  wholesome.  Why,  then,  make  sanitar\-  analyses 
to  determine  the  presence  of  sewage  ?     If  it  be  contended  that  this 


f 


52  Marshall  O.  Leighton 

area  may  be  subject  to  occasional  malicious  pollutions  by  visitors, 
etc.,  then  the  sanitary  analysis  does  not  offer  any  helpful  solution. 
A  single  infectious  intestinal  discharge  deposited  directly  in  a  reser- 
voir might  readily  cause  a  typhoid  epidemic,  but  the  organic  matter 
would  not,  except  under  very  fortunate  circumstances,  be  detected 
by  the  nitrogen  determinations;  and  it  is  well  to  reflect  that,  under 
the  usual  conditions,  by  the  time  the  disease  had  made  itself  manifest 
and  attention  directed  to  that  reservoir,  the  infection  would  have  passed 
out  of  the  reservoir. 

If  the  drainage  area  above  described  does  contain  population, 
then  the  danger  is  always  impending,  and  we  may  rest  upon  assump- 
tions, nearly,  if  not  quite,  as  positive  as  those  quoted  above  for  river 
waters.  It  is  quite  significant  in  this  connection  to  note  that  the 
Commission  on  Additional  Water  Supply  of  the  City  of  New  York 
made  provision  for  filtration,  although  the  upland  areas  proposed 
as  new  sources  are  sparsely  settled.  More  recently  we  have  read 
the  opinions  of  our  foremost  authorities  that  the  present  Croton 
supply  should  be  filtered.  It  is  doubtful  if  any  of  those  authorities 
would  contend  that  the  sole  object  of  such  filtration  is  to  remove 
turbidity,  color,  and  odor.  It  is  therefore  held  that  the  sanitary 
analysis  of  upland  conserved  supplies  is  needless,  because  we  can 
determine  the  danger  by  inspection  far  more  readily  and  surely. 

With  reference  to  ground  waters :  We  have  interesting  accounts 
of  cases  in  which  it  is  asserted  that  the  condition  of  pollution  was 
not  detected  by  biological  examination,  but  was  revealed  by  sanitary 
analysis.  If  close  consideration  be  given  to  the  descriptions  of 
premises  that  appear  in  these  accounts,  it  will  be  seen  that  in  every 
case  (so  far  as  the  writer  is  informed)  a  careful  man  would  have 
been  justified  in  condemning  those  waters  upon  superficial  examina- 
tion, and  without  regard  to  analysis.  Take  for  illustration  the  case 
cited  by  Professor  WiUiam  P.  Mason  in  a  paper  entitled,  "  Interpre- 
tation of  a  Water  Examination,"  which  appeared  in  Science^  Vol.  21, 
No.  539,  pp.  648-53.  It  appears  from  this  that  there  was  a  certain 
farmhouse  in  England,  the  residents  of  which  had  suffered  severely 
from  diphtheria  and  typhoid  fever.  Examination  showed  that  the 
sewage  discharged  from  the  house  entered  into  a  dry-steyned  cess- 
pool, without  overflow,  about  four  yards  from  the  well,  both  sunk 


Futility  of  a  Sanitary  Water  Analysis  53 

in  gravel.  In  this  case  the  chemical  examination  revealed  the  pres- 
ence of  an  excess  of  chlorides  and  nitrates  while  bacteriological 
investigation  showed  nothing  which  would  cause  suspicion.  This 
instance  is  cited  by  Professor  Mason  as  one  in  which  "the  danger 
signal  was  held  out  by  the  chemical  side  of  the  investigation  alone." 
The  writer  is  of  the  opinion  that  the  "danger  signal"  was  not  the 
findings  of  the  analysis,  but  the  occurrence  of  disease  in  that  residence. 
It  should  have  caused  an  immediate  examination  of  the  premises, 
and  such  examination  would  have  revealed  the  fact  that  the  sewage 
was  discharged  into  a  dry-steyned  cesspool,  that  the  well  was  only 
jour  yards  away  and  that  both  were  sunk  in  gravel.  In  the  face  of  all 
our  knowledge  of  the  transmission  of  water-borne  diseases,  and  in 
view  of  our  decades  of  experience  with  infected  wells,  from  historic 
Broad  Street  down  to  the  present,  why  should  any  competent  obser- 
ver, with  the  above  related  facts  before  him,  find  it  necessary  to 
fuss  with  an  ammonia  still  or,  for  that  matter,  with  a  Petri  dish? 
Taking  a  broad  view  of  the  subject  of  well  supplies,  we  may  safely 
exclude  all  wells  in  questionable  places;  and  the  careful  observer 
can  usually  define  such  places. 

All  of  the  above  discussion  with  reference  to  the  needlessness 
of  sanitary  analyses,  when  other  and  more  expeditious  methods 
can  be  used,  is  based  upon  the  temporar)^  admission  that  such  anal- 
yses afford  data  whereby  dangerous  animal  pollution  can  be  dis- 
tinguished from  harmless  vegetable  matter.  If  we  return  now  to 
the  original  contention  that  standards  of  purity,  bases  of  interpreta- 
tion, composition  ratios,  or  by  whatever  name  they  may  be  called, 
are  met  with  equal  faith  by  the  normal  water  and  by  the  dilute  sewage, 
and  sum  up  the  two  lines  of  evidence,  we  have  what  the  writer  feels 
justified  in  regarding  as  an  established  case  against  the  sanitary 
analysis  as  an  index  of  dangerous  water  pollution. 


V 


THE  VALUE   OF   PURE   WATER* 

George  C.  Whipple. 
PREFACE. 

In  order  to  estimate  the  relative  value  of  waters  which  differ 
materially  in  quality,  it  is  necessary  to  have  some  common  denomi- 
nator. Nothing  better  for  this  purpose  has  been  suggested  than 
the  dollar,  which  in  this  paper  is  made  the  basis  of  computation.  By 
ascertaining  what  different  characteristics  of  water  cost  the  con- 
sumers, and  by  finding  out  how  much  consumers  are  willing  to 
pay  to  avoid  using  waters  which  possess  certain  characteristics,  an 
attempt  has  been  made  to  secure  a  reasonable  basis  of  comparison. 
The  results  of  this  initial  study  are  here  presented.  They  must 
not  be  taken  too  seriously  at  present,  as  some  of  the  involved  as- 
sumptions have  not  been  established  beyond  doubt;  and  with  the 
accumulation  of  certain  data,  necessary  but  not  as  yet  obtainable, 
the  results  must  be  somewhat  modified.  Yet  the  general  conclu- 
sions ought  not  to  be  far  astray,  and,  from  a  study  of  the  best  data 
available,  the  writer  believes  that  they  err  on  the  side  of  conserva- 
tism rather  than  on  the  opposite  side.  The  suggested  method  of 
calculating  the  value  of  pure  water  seems  to  be  one  capable  of  being 
refined  to  a  degree  where  its  results  will  be  of  great  practical  value. 
The  lines  along  which  the  accumulation  of  data  is  necessary  in  order 
to  render  the  method  reliable  will  be  evident  from  a  perusal  of  the 
text. 

PURE  AND  WHOLESOME  WATER. 

To  define  the  meaning  of  the  expression  "pure  and  wholesome 
water,"  which  is  so  often  found  in  water-supply  contracts,  would 
seem  to  be  an  easy  matter,  after  all  the  study  that  has  been  given 
to  the  subject  in  recent  years;  but,  although  everyone  knows  in  a 
general  way  what  is  implied  by  this  expresssion,  yet  when  it  comes 
to  framing  a  definition  in  positive  scientific  terms,  the  problem  is  not 
as  easy  as  it  seems.  This  is  not  because  the  chemist  and  the  biolo- 
gist do  not  know  what  pure  water  is,  but  because  water  has  so  many 

♦Received  for  publication  February  17,  1906. 

54 


The  Value  of  Pure  Water  55 

attributes  which  have  to  be  taken  into  consideration,  and  because 
these  attributes  vary  in  importance  in  every  instance.  "Pure  and 
wholesome  water"  is  not  a  substance  of  absolute  quality.  Strictly 
speaking,  pure  water  does  not  exist  in  nature;  all  natural  waters 
contain  substances  either  in  solution  or  suspension;  and  in  propor- 
tion as  these  substances  are  present,  and  in  proportion  as  they  are 
objectionable  in  character,  the  water  is  impure.  Definitions  of  pure 
and  wholesome  water,  therefore,  generally  state  what  foreign  sub- 
stances shall  not  be  present,  or  in  what  amounts  they  are  permis- 
sible, instead  of  defining  the  positive  qualities  which  the  water  shall 
possess. 

Unquestionably  the  term  "pure  and  wholesome  water,"  as  ordi- 
narily used,  relates  to  water  intended  to  be  used  for  drinking.  Such 
a  water  must  be  free  from  all  poisonous  substances,  as  the  salts  of 
lead ;  it  must  be  free  from  bacteria  or  other  organisms  liable  to  cause 
disease,  such  as  the  bacilli  of  typhoid  fever  or  dysentery;  it  must 
also  be  free  from  bacteria  of  fecal  origin,  such  at  B.  coli.  In  other 
words,  the  water  must  be  free  from  poisonous  substances,  from 
infection,  and  even  from  contamination,*  Besides  this,  it  must  be 
practically  clear,  colorless,  odorless  and  reasonably  free  from  objection- 
able chemical  salts  in  solution  and  from  microscopic  organisms  in 
suspension.  Moreover,  it  must  be  well  aerated.  Color,  turbidity, 
odor,  dissolved  salts,  etc.,  may  be  permissible  to  a  small  degree  with- 
out throwing  the  water  outside  of  the  definition  of  pure  and  whole- 
some waters.  In  these  minor  matters  local  standards  govern  up  to 
a  certain  point,  and  it  is  in  regard  to  them  that  differences  in  the 
judgment  and  experience  of  analysts  lead  to  different  classifications. 

When  it  comes  to  using  water  for  other  purposes  than  for  drinking, 
other  attributes  have  to  be  considered.  Hardness  makes  a  water 
troublesome  to  wash  with  and  to  use  in  boilers;  iron  makes  trouble  in 
the  laundry;  chlorine  corrodes  pipes  and  makes  work  for  the  plumb- 
ers; the  presence  of  the  carbonates  and  sulphates  of  lime  and  mag- 
nesia affects  the  paper-maker,  the  brewer,  the  tanner,  the  dyer,  the 
bleacher;  soda  causes  a  locomotive  boiler  to  foam,  and  affects  the  use 
of  the  water  for  irrigation.     All  of  these  constituents,  and  others  which 

♦By  this  term  is  meant  pollution  with  fecal  matter.  Contamination  must  be  considered  as  potential 
infection. 


56  George  C.  Whipple 

are  not  named,  have  to  be  taken  into  consideration  in  connection  with 
a  public  water  supply,  w^hich  may  be  put  to  any  of  these  uses. 

If  it  is  a  difficult  matter  to  define  a  pure  and  wholesome  water  in 
strict  scientific  terms,  it  is  still  more  difficult  to  compare  waters  which 
differ  in  purity  on  any  reasonable  basis;  and  yet  this  often  has  to  be 
done.  Given  two  water  sources  equally  available  to  a  city  for  pur- 
poses of  supply,  both  safe  to  drink,  but  one  high-colored  and  soft,  the 
other  colorless  and  hard — which  is  the  better  selection  ?  A  water- 
works plant  is  to  be  appraised :  structurally  the  system  is  a  good  one 
but  the  quality  of  the  water  is  unsatisfactory  because  of  its  excessive 
color  or  turbidity — how  much  should  be  deducted  from  the  value  of 
the  works  because  of  the  bad  quality  of  the  water  ?  The  water- works 
owned  by  a  private  company  are  to  be  purchased  by  the  city ;  the  city 
has  a  high  typhoid  fever  death-rate  due  unquestionably  to  the  water 
supply — how  much  less  should  the  city  pay  because  of  that  fact  ?  A 
city  in  the  West  is  using  turbid  river  water — how  much  can  it  afford 
to  pay  to  filter  it  ?  A  city  in  New  England  is  using  a  water  so 
heavily  laden  with  Anabaena  that  it  is  nauseous  to  drink — how  much 
can  the  city  afford  to  pay  to  procure  a  new  supply  ?  These  are  all 
practical,  everyday  questions  which  deserve  answers  based  on  scien- 
tific data. 

In  valuation  cases,  where  the  quality  of  the  water  supply  has  been 
unsatisfactory,  the  cost  of  filtration,  or  other  appropriate  method  of 
purification,  has  been  sometimes  taken  as  a  measure  of  the  inferior 
quality  of  the  water,  and  this  amount  deducted  from  the  value  of  the 
works.  In  case  filtration  was  impractical,  or  more  expensive  than 
securing  a  supply  from  a  new  source,  the  additional  cost  of  such  new 
supply  has  been  sometimes  taken  as  a  measure  of  the  inferior  quality 
of  the  works  and  the  amount  deducted  from  the  value  of  the  works. 
Both  of  these  methods  are  similar  in  that  they  contemplate  the  sub- 
stitution of  a  satisfactory  water  for  one  not  satisfactory. 

Another  method  of  measuring  the  depreciation  applicable  to  a 
water-works  plant  because  of  an  inferior  quality  of  the  supply 
would  be  to  ascertain  what  the  use  of  the  impure  water  has  cost  the 
consumers,  compared  with  what  a  pure  and  satisfactory  water  would 
have  cost  them.  This  method  has  not  been  used  in  practice,  but  it 
seems  to  be  a  reasonable  one,  and  one  which  would  be  of  more  general 


The  Value  of  Pure  Water  57 

application  than  the  preceding,  if  the  data  upon  which  it  is  based 
could  be  accurately  determined.  Unfortunately,  this  is  not  the  case 
in  many  instances,  but  by  the  use  of  certain  generalized  data  and 
assumptions,  results  may  be  secured  which  are  of  considerable  use  in 
comparing  the  value  of  waters  different  in  quality. 

The  qualities  of  a  public  water  supply  which  most  affect  the  ordi- 
nary consumer  are : 

1.  Its  sanitary  quality;  that  is,  its  liability  of  infection  with  disease 
germs  or  substances  deleterious  to  health. 

2.  Its  general  attractiveness,  or  lack  of  attractiveness,  as  a  drinking- 
water. 

3.  Its  hardness,  so  far  as  this  relates  to  the  use  of  soap  in  the 
household. 

4.  Its  temperature,  so  far  as  this  relates  to  drinking. 
Characteristics  which  affect  industrial  uses  are  too  much  a  matter 

of  local  concern  to  be  taken  into  account  in  a  general  discussion, 
although  they  are  by  no  means  of  small  account,  and  in  some  com- 
munities their  importance  might  control.  The  qualities  selected  are 
to  be  considered  as  illustrative  of  the  method  rather  than  as  a  com- 
plete exposition  of  it. 

The  problem  is  to  express  these  four  characteristics  in  terms  of 
dollars  and  cents  to  the  consumer.  The  financial  standard  is  cer- 
tainly not  the  highest  one  for  judging  the  quality  of  a  water  supply 
when  the  public  health  is  concerned ;  human  life  cannot  be  estimated 
in  gold  dollars,  and  the  smell  of  unsavory  water  to  a  thirsty  man  cannot 
be  reckoned  in  dimes;  nevertheless,  the  financial  basis  is  a  convenient 
one,  and  one  necessarily  involved  in  all  questions  which  relate  to  public 
utilities. 

SANITARY  QUALITIES. 

If  the  water  under  consideration  has  been  used  for  a  considerable 
time,  the  typhoid  fever  death-rate  of  the  community  will  fairly  well 
represent  the  sanitary  quality  of  the  water  supply.  It  will  not  tell 
the  whole  story,  but  in  most  cases  it  will  not  lead  far  astray.  In  order 
to  reduce  this  to  a  financial  basis,  it  is  necessary  to  make  several 
assumptions. 

The  financial  value  of  a  human  life  is  generally  taken  as  $5,000, 


58 


George  C.  Whipple 


but  according  to  Leighton'  it  varies  at  different  ages  from  $i,ooo  to 
$7,000,  as  shown  by  Table  i.  It  so  happens  that  persons  are  most 
susceptible  to  typhoid  fever  near  the  age  when  their  life-value  is 
considered  greatest.  By  combining  the  life-value  at  different  ages 
with  the  age  distribution  of  persons  dying  of  typhoid  fever,  the 
resulting  average  value  of  persons  dying  from  typhoid  fever  is  found 
to  be  $4,634,  which  is  very  close  to  the  figure  ordinarily  used. 

The  percentage  mortality  of  typhoid  fever  patients  is  sometimes 
stated  as  10  per  cent;  that  is,  ten  cases  for  every  death.  Figures  of 
this  character  are  most  often  based  on  hospital  records,  and  mild  cases 
do  not  generally  reach  the  hospitals.  Studies  of  recent  typhoid  epi- 
demics indicate  that  15  to  18  cases  for  each  death  would  be  nearer  the 
truth.  The  expense  of  medical  treatment,  nursing,  and  medicine,  the 
loss  of  wages  for  a  month  or  more,  together  with  other  attending 
expenses  and  inconveniences,  would  doubtless  aggregate  at  least  $100 
per  case,  or  $1,000  for  the  10  cases  corresponding  to  one  death.  If 
the  estimate  of  $100  is  considered  too  large,  it  may  be  answered  that 
the  excess  is  more  than  offset  by  the  fact  that  there  are  more  often  from 
15  to  18  cases  for  each  death  than  there  are  10.  It  may  be  fairly 
assumed,  therefore,  that  $6,000  is  a  very  moderate  estimate  of  the 
financial  loss  to  the  community  from  typhoid  fever  for  each  death 
from  that  disease. 

TABLE  I. 


Age 
o-  s  years 

s-°  ::  

10-15  

15-20  "  

20-2S  "  

25-30  "  

30-35  '  

35-4°  ;;  

40-45      

4S-50  :: 

50-55  

SS-60     "      

60-65     "      

65-70     "      

70-  

Total 


Estimated  Value  of 
Human  Life 


$1. 
2 
2 
3 
5' 
7 
7 
6 

5 
5 
4 
4 
2 
I 
I 


500 
300 
500 
000 
000 
500 
,000 
000 
500 
,000 
Soo 
500 
,000 
,000 
,000 


Per  Cent  of  Deaths 
from  Typhoid  Fever 


5 

5 

7 

13 

16 

13 
9 
8 

S 
4 
3 
2 
2 
I 
I 


Product  of  Columns 
2  and  3 


$  7-5 10 

13.570 

18,000 

39.300 

83.500 

99,100 

60,300 

48,000 

30,900 

20,000 

15,000 

11,700 

4,200 

1.500 

1,900 


$463,480 


Average  value  of  life  of  persons  dying  from  typhoid  fever,  $4,634. 
'M.  O.  Leighton,  Popular  Science  Monthly,  January,  1902. 


The  Value  of  Pure  Water 


59 


TABLE  2. 

Effect  of  FatRAXiON  on  Death-Rates  at  Albany,  N.  Y.,  and  a  Comparison  with  Troy,  N.  Y., 

Where  the  Water  Was  Not  Filtered. 


Death-Rates  per  100,000 


1804-98,  before 
Filtration 
at  Albany 


I 900- I 004 

after  Filtration 

at  Albany 


Difference 


Per  Cent  Re- 
duction of 
Death  Rates 


Albany 

Typhoid  fever 

104 

125 

606 
2.264 

26 

53 

309 

1,868 

78 

72 

297 

378 

75 

Diarrheal  diseases 

57 

Children  under  5  years 

49 

Total  deaths 

17 

Troy 

Typhoid  fever  

57 
116 

531 

2,157 

57 
102 

2,028 

0 
14 
06 

120 

0 

Diarrheal  diseases 

12 

Children  under  5  years 

18 

Total  deaths   ... 

6 

Remark:  Filtered  water  was  introduced  into  Albany  in  1899. 
of  Troy  has  remained  practically  unchanged. 


The  water  supply 


Typhoid  fever  is  by  no  means  the  only  disease  transmitted  by 
contaminated  water.  Dysentery  and  various  other  diarrheal  diseases 
precede  it  or  follow  in  its  train,  and  in  most  instances  these  are  prob- 
ably due  to  the  same  general  sources  of  contamination  as  those  w^hich 
caused  the  typhoid  fever,  although,  of  course,  to  different  specific 
infections.  The  reduction  of  the  typhoid  fever  death-rate  following 
the  substitution  of  a  pure  water  for  a  contaminated  water  is  often 
accompanied  by  a  drop  in  the  death-rate  from  other  diseases.  Thus, 
if  the  five  years  before  and  after  filtered  water  was  introduced  into 
Albany,  N.  Y.,  are  compared,  it  will  be  seen  that  the  reductions  in 
deaths  from  general  diarrheal  diseases  and  the  deaths  of  children 
under  five  years  of  age  were  much  greater  than  in  the  case  of  typhoid 
fever.  There  was  also  a  reduction  in  malaria,  but  this  probably 
represents  faulty  diagnosis  of  typhoid  fever  cases  before  the  introduc- 
tion of  the  filters,  rather  than  a  real  reduction  of  malaria.  That  the 
reduction  of  infant  mortality  and  deaths  from  diarrheal  diseases  was 
not  due  to  other  conditions  seems  probable  from  the  fact  that  in  the 
neighboring  city  of  Troy,  where  the  water  supply  was  not  changed, 
there  was  no  such  diminution  during  the  same  period.     (Sec  Table  2.) 

Hazen,  in  his  paper  on  "Purification  of  Water  in  America,"  read 


6o  George  C.  Whipple 

at  the  International  Engineering  Congress  at  St.  Louis,  called  atten- 
tion to  this  same  fact,  that  after  the  change  from  an  impure  to  a  pure 
supply  of  water  the  general  death-rate  of  certain  communities  investi- 
gated fell  by  an  amount  considerably  greater  than  that  resulting  from 
typhoid  fever  alone — indicating  either  that  certain  other  infectious 
diseases  were  reduced  more  than  typhoid  fever,  or  that  the  general 
health  tone  of  the  community  had  been  improved.  Thus,  for  five 
cities  where  the  water  supply  had  been  radically  improved  he  found  : 

Per  icx3,ooo 
Reduction  in  total  death-rate  in  five  cities  with  the  introduction  of  a  pure  water 

supply 440 

Normal  reduction  due  to  general  improved  sanitary  conditions,  computed  from 

average  of  cities  similarly  situated,  but  with  no  radical  change  in  water  supply      137 

Difference,  being  decrease  in  death-rate  attributable  to  change  in  water  supply  .  303 
Of  this,  the  reduction  in  deaths  from  typhoid  fever  was         71 

Leaving  deaths  from  other  causes  attributable  to  change  in  water  supply     .     .     .     232 

From  these  facts  it  is  evident  that  to  place  the  financial  loss  to  a 
community  as  $6,000  for  each  death  from  typhoid  fever  due  to  the 
public  water  supply  is  to  use  too  low  a  figure.  It  probably  ought  to  be 
several  times  as  high ;  but  recognizing  the  lower  financial  value  placed 
on  the  lives  of  infants,  and  the  less  serious  character  of  the  other  dis- 
eases, and  wishing  to  be  as  conservative  as  possible,  for  the  reason 
that  typhoid  fever  is  not  entirely  a  water-borne  disease,  $10,000  per 
typhoid  death  has  been  used  in  the  calculation  which  follows. 

Since  typhoid  fever  is  a  disease  which  may  be  transmitted  in  other 
ways  than  by  the  water  (as,  for  instance,  by  milk,  shell-fish,  or  flies),  it 
is  necessary  to  allow  a  certain  death-rate  for  these  other  causes,  for 
even  in  a  city  where  the  water  supply  is  perfect  there  may  still  be  some 
typhoid  fever.  To  establish  this  "normal"*  is  a  difficult  matter,  but 
for  purposes  of  calculation  we  may  assume  it  to  be  determined  and 
represent  it  by  the  letter  N. 

If  we  assume  that  all  typhoid  fever  in  excess  of  N  is  due  to  the 
water  supply,  and  if  we  assume  that  the  daily  consumption  of  water  is 
100  gallons  per  capita,  then  letting  T  equal  the  typhoid  fever  death- 
rate  per  100,000 — 

(T—N)  10,000  =  loss  to  the  community  in  dollars  for  365 X 100 X 

11           r       ^             T>     {T-N)i,ooo 
100,000  gallons  of  water,  or  D= 7 ■  =2.75(7  —A'), 

♦This  term  "normal"  must  not  be  assumed  to  mean  necessary  typhoid. 


The  Value  of  Pure  Water 


6i 


where  D  stands  for  the  loss  in  dollars  per  million  gallons  of  water 
used. 

Suppose  the  average  typhoid  fever  death-rate  for  a  community 
which  has  a  somewhat  polluted  water  supply  has  averaged  43  per 
100,000  for  a  period  of  five  years,  and  suppose  that  for  this  place  the 
value  of  A''  is  estimated  as  15,  then — 

Z)  =  2.75  (43—15)  ~  $76.72  if  the  per  capita  consumption  is  100 
gallons.  If  the  consumption  per  capita  is  115  gallons,  D  would  be 
W^  of  $76.72,  or  $66.71;  if  it  were  63  gallons  per  capita,  then  D 
would  equal  ^^^  of  $76. 72,  or  $121 . 77. 

The  value  of  N  must  be  naturally  subject  to  local  variation,  and  in 
order  to  obtain  an  idea  as  to  its  probable  value,  a  compilation  of 
typhoid  fever  death-rates  has  been  made  for  cities  and  towns  in  differ- 
ent parts  of  the  country  which  use  ground  waters  or  filtered  waters — 
that  is,  waters  which  may  be  considered  as  free  from  contamination. 

The  following  is  a  generalized  summary  of  them : 

TABLE  3. 
Typhoid  Fever  Death-Rates  m  Cities  and  Towns  Which  Have  Ground- Water  Supplies. 


State 


Maine 

Massachusetts 
Connecticut  . . 
New  York  . .  . 
New  Jersey  . . 
Pennsylvania. 
Ohio 


Number  of  Cities 

and  Towns 

Averaged 


2 
23 

4 
I3 
10 

s 

22 


Number  of  Years 
Averaged 


Average  Typhoid 

Fever  Death- Rate 

per  100.000 


6.4 
15-8 

9-5 
24.7 
20.5 
31.8 
32.4 


There  is  reason  to  believe  that  the  higher  rates  given  above  do 
not  correctly  represent  the  situation,  because  in  some  instances  the 
ground  water  was  supplemented  by  the  occasional  use  of  water  which 
may  have  been  polluted.  Proximity  to  a  large  city  where  the  water 
supply  is  contaminated  was  also  responsible  for  some  of  the  high 
figures;  so  also  was  the  absence  of  sewerage  systems.  Nevertheless, 
there  seems  to  be  a  slight  tendency  for  the  typhoid  fever  rates  to 
increase  in  the  United  States  from  north  toward  the  south  in  those 
places  where  the  water  supply  is  reasonably  safe.  There  are  some 
exceptions  to  the  increase  southward,  however.  Thus,  in  Camden, 
N.  J.,  which  is  supplied  with  a  pure  ground  water,  the  typhoid  rate 
in  1901  was  only  12,  and  20  in  1902. 


62  George  C.  Whipple 

In  Fuertes'  book  on  Water  and  the  Public  Health  sl  diagram  is 
given  showing  that  the  typhoid  fever  death-rates  in  cities  supphed 
with  ground  water  vary  from  5  to  32  per  100,000  in  America,  and  from 
6  to  T)T,  per  100,000  in  Europe,  the  average  being  about  18  in  America 
and  19  in  Europe.  It  is  shown  also  that  the  death-rates  from  cities 
supphed  with  fiUered  water  vary  from  4  to  20  in  America,  and  from 
4  to  20  in  Europe,  the  average  being  12  in  both  cases.  Recent  Ameri- 
can data  for  cities  supphed  with  fiUered  water  show  that  the  rates  are 
somewhat  higher  than  these,  the  average  being  somewhat  less  than  20. 

Taking  into  consideration  the  best  available  data,  it  seems  reason- 
able to  place  the  general  value  of  A^  somewhere  between  10  and  25 
per  100,000,  with  the  most  probable  average  value  as  20,  which  figure 
may  be  used  in  the  equation  where  local  sanitary  conditions  are 
unknown.  The  value  of  N,  however,  should  be  varied  where  there  is 
reason  for  doing  so.  Where  the  sanitary  conditions  are  good  15  may 
be  taken  as  a  fair  value.  In  New  England  it  might  be  placed  lower 
than  in  regions  south  of  the  glacial  drift ;  in  cities  near  the  seaboard, 
where  there  is  a  large  consumption  of  oysters  consumed  fresh  from 
the  layings,  the  value  of  N  might  be  higher  than  in  inland  cities, 
where  the  oyster  consumption  is  small  and  where  fattened  oysters  are 
not  used  as  freely;  in  cities  where  there  are  cess-pools,  but  no  sewers, 
the  value  of  N  would  naturally  be  higher  than  in  cities  well  provided 
with  sewers. 

It  may  be  reasonably  expected  that,  as  time  goes  on,  the  value  of 
N  will  gradually  fall,  because  of  a  general  decrease  of  typhoid  fever  in 
the  country  at  large,  and  a  consequent  diminution  of  the  number  of 
foci  of  infection.  Statistics  for  twelve  states,  including  all  the  New 
England  states.  New  York,  New  Jersey,  Maryland,  California,  Min- 
nesota, and  Michigan,  show  that  during  the  last  quarter  of  a  century 
the  general  typhoid  fever  death-rate  has  fallen  as  follows: 

TABLE  4 

Average  Typhoid 
Fever  Death- 
Rate  per 
Year  100,000 

1880 55 

1885 46 

1890 36 

1895 28 

1900 23 

1905 21 


The  Value  of  Pure  Water 


63 


ATTRACTIVENESS. 

The  analytical  determinations  which  relate  to  the  general  attrac- 
tiveness of  a  water  are  those  of  taste,  odor,  color,  turbidity,  and  sedi- 
ment. As  these  quantities  increase  in  amount,  the  water  becomes 
less  attractive  for  drinking  purposes,  until  finally  a  point  is  reached 
where  people  refuse  to  drink  it.  In  order  to  use  these  results  in  a 
practical  way,  it  is  necessary  to  combine  them  so  as  to  obtain  a  single 
value  for  the  physical  characteristics  or,  as  they  say  abroad,  for  the 
"organoleptic"  quality  of  the  water.  An  attempt  has  been  made  by 
the  author  to  obtain  what  may  be  termed  an  esthetic  rating  of  the 
water,  and  the  result  is  shown  in  the  accompanying  diagram. 


I  X  <i  ij 


Tu^eiD/Tf  jif^c  coj-off     (^jatrrj    ^jr^    AJ/j-^'On/J 


This  diagram,  it  should  be  said,  is  based  almost  entirely  upon  esti- 
mates and  very  little  upon  statistical  data.  It  rests  upon  the  assump- 
tion that  people  differ  in  their  sensibilities,  or  their  esthetic  feelings  as 
to  the  use  of  water.  Some  persons  are  much  more  fastidious  than 
others  in  regard  to  what  they  drink.  A  water  which  would  be  shunned 
by  one  person,  even  though  he  were  thirsty,  might  be  taken  by  another 
with  apparent  relish.  As  a  rule,  people  are  more  fastidious  about  the 
odor  of  water  and  the  amount  of  coarse  sediment  which  it  contains 
than  they  are  about  its  color  and  turbidity.  This  is  perhaps  natural, 
as  a  bad  odor  suggests  decay,  and  decay  is  instinctively  repugnant. 


64  George  C.  Whipple 

Often,  however,  people  do  not  discriminate  between  odors  which  are 
due  to  decomposition  and  those  which  are  not.  Habit  and  associa- 
tion have  much  to  do  with  a  person's  views  as  to  the  attractiveness  of 
water.  In  New  England,  where  the  clear  trout  brooks  run  with  what 
Thoreau  called  "meadow  tea,"  few  people  object  to  a  moderate 
amount  of  color,  while  they  do  object  to  a  water  which  is  very  turbid. 
In  the  Middle  West,  where  all  the  streams  are  muddy,  it  is  the  un- 
known colored  waters  which  are  disliked.  People  who  are  accus- 
tomed to  well  water  object  to  both  color  and  turbidity.  With  most 
people  a  fine  turbidity,  such  as  is  produced  by  minute  clay  particles, 
is  less  a  subject  of  complaint  than  an  equal  turbidity  produced  by 
comparatively  coarse  sediment.  In  the  diagram  an  attempt  has  been 
made  to  reconcile  these  different  points  of  view  so  as  to  put  them,  as 
weU  as  may  be,  on  the  same  footing.  In  this  connection  several  series 
of  comparisons  were  made.*  Turbid  waters  were  viewed  through 
the  eyes  of  a  group  of  western  people,  who  made  some  comparisons 
with  color  and  turbid  waters,  while  colored  waters  were  viewed  through 
the  eyes  of  a  group  of  students  in  New  York,  and  vice  versa. 

The  abscissae  of  the  diagram  represent  turbidity,  color,  and  odor, 
as  given  in  the  ordinary  water  analysis. f  The  ordinates  represent  the 
"per  cent  of  objecting  consumers."  By  this  is  meant  the  proportion 
of  the  water-takers  who  would  ordinarily  choose  not  to  drink  the  water 
because  of  the  quality  indicated  by  the  curve,  or  who  would  buy  spring 
water,  or  bottled  water,  rather  than  use  the  public  supply,  if  they 
could  afford  to  do  so.  This  number  would  increase,  of  course,  as  the 
general  attractiveness  of  the  water  decreased.  From  the  curves  one 
may  calculate  what  may  be  called  the  esthetic  deficiency  of  the  water 
by  adding  together  the  per  cents  of  objecting  consumers  for  color, 
turbidity,  and  odor.  If  the  esthetic  deficiency  equals  loo,  it  indicates 
that  the  water  is  of  such  a  character  that  everyone  would  object  to  it, 
and  figures  in  excess  of  loo  only  emphasize  its  objectionable  character. 

It  will  be  seen  from  the  diagram  that  when  the  color  of  water  is 
less  than  20,  or  the  turbidity  less  than  5,  only  one  person  in  ten  would 
object  to  it,  but  when  the  turbidity  or  color  is  100,  one-half  of  the 

♦Acknowledgments  are  due  to  Mr.  J.  W.  Ellms,  of  Cindnnati,  Ohio,  and  Mr.  Andrew  Mayer,  Jr., 
of  Brooklyn,   N.  Y. 

tSee  "  Report  of  Committee  on  Standard  Methods  of  Water  Analysis,  American  Public  Health  Asso- 
ciation,"  Supplement  No.  i.  Journal  of  Infectious  Diseases,  May,  1905. 


The  Value  of  Pure  Water  65 

people  would  object  to  it.  It  may  be  thought  that  this  proportion  is 
too  low,  but  it  must  be  remembered  that  colored  waters  are  invariably 
accompanied  by  a  vegetable  odor  and  often  by  a  slight  turbidity,  and 
that  it  is  the  sum  of  the  several  quantities  which  determines  the  esthe- 
tic rating. 

Experience  has  shown  that  objection  to  color  varies  directly  with 
its  amount ;  consequently  this  curve  has  been  plotted  from  the  equa- 
tion, pc  =  —  ,    i.  e.,  a  straight  line,  where  pc  stands  for  the  per  cent  of 

objecting  consumers,  and  c  for  the  color. 

In  the  case  of  turbidity,  however,  small  amounts  count  for  more, 
relatively,  than  larger  amounts.  The  equation  for  the  turbidity  curve 
has  been  taken,  therefore,  a,s  pi  =  $V t,  where  t  stands  for  the  turbidity. 

With  odor,  however,  the  opposite  condition  prevails;  faint  odors 
count  for  little,  but  distinct  and  decided  odors  cause  much  more  com- 
plaint. Consequently,  the  per  cent  of  objecting  consumers  has  been 
made  to  vary  as  the  square  of  the  intensity  of  the  odor  expressed 
according  to  the  standard  numerical  scale.  The  quality  of  the  odor 
makes  quite  as  much  difference  as  its  intensity,  and  for  that  reason 
three  curves  have  been  plotted,  one  representing  vegetable  or  pondy 
odors  (Oj,),  one  representing  odors  due  to  decomposition  (Oj),  and 
one  representing  the  aromatic  grassy  and  fishy  odors  due  to  micro- 
scopic organisms  (Og).  These  curves  are  plotted  from  the  following 
equations : 

p.=5o:, 

Po=20l, 

in  which  O^,  Od,  and  Oy  stand  for  the  intensity  of  the  three  groups  of 
odors  mentioned. 

These  curves  represent  somewhat  imperfectly  our  present  ideas  as 
to  the  relative  effects  of  color,  turbidity,  and  odor;  and  on  further 
study  they  are  likely  to  be  considerably  modified. 

It  is  a  well-know^n  fact  that  in  cities  which  are  supplied  with  water 
which  is  not  attractive  for  drinking  purposes,  large  quantities  of 
spring  water  and  distilled  water  are  sold,  and  that  consumers  go  to 
much  expense  in  the  purchase  of  house  filters  in  order  to  improve  the 
quality  of  the  water  furnished  by  the  city  mains.     It  is  fair  to  assume 


66  George  C.  Whipple 

that  in  any  community  the  amount  of  money  expended  for  bottled 
water  and  house  filters  will  vary  in  a  general  way  according  to  the 
attractiveness  of  the  water,  although  there  is  no  doubt  that  the  presence 
of  typhoid  fever  in  the  community,  or  the  fear  that  the  water  is  con- 
taminated, will  greatly  increase  the  use  of  auxiliary  supplies  for  drink- 
ing. For  purposes  of  calculation  it  may  be  assumed  that  the  diagram 
just  described  represents  this  tendency  to  use  vended  waters,  and  that 
each  "objecting  consumer"  would  go  to  the  expense  of  buying  spring 
water  or  putting  in  a  house  filter,  if  he  could  afford  it.  It  may  be 
argued,  also,  that  the  poor  consumer  who  may  be  unable  to  do  this 
is  as  much  entitled  to  satisfactory  water  as  is  the  well-to-do  consumer. 

From  a  study  of  price-lists  of  spring  waters  sold  in  New  York  and 
other  cities  it  has  been  found  that  the  ordinary  wholesale  price  of 
spring  water  is  seldom  more  than  lo  cents  a  gallon.  In  some  places  it 
is  as  low  as  i  cent.  The  average  is  about  5  cents.  To  filter  water 
through  house  filters  costs  less,  but  generally  it  is  less  satisfactory. 

As  a  convenient  figure  for  calculation,  and  as  a  most  conservative 
one  for  general  use,  a  cost  of  i  cent  per  gallon  to  the  ordinary  con- 
sumer for  an  auxiliary  supply  of  drinking-water  (either  spring  water 
or  well-filtered  water)  has  been  taken.  In  cities  where  the  cost  of 
procuring  and  distributing  bottled  water  exceeds  i  cent  per  gallon, 
as  it  does  in  such  a  city  as  New  York  for  example,  this  should  be  taken 
into  account  in  making  local  use  of  the  data.  For  the  illustrative  pur- 
poses of  the  present  paper,  and  for  general  comparisons,  the  figure 
mentioned  will  serve  as  a  satisfactory  basis.  The  average  person 
drinks  about  i .  5  quarts  of  water  per  day,  and  therefore  one-fifth  cent 
per  capita  daily  may  be  taken  as  a  reasonable  figure  for  the  cost  of  an 
auxiliary  supply.  If  the  entire  population  used  such  a  supply,  and  if 
the  daily  consumption  of  the  public  water  supply  were  100  gallons  per 
capita,  then  one-fifth  cent  per  hundred  gallons,  or  $20  per  million 
gallons,  would  represent  the  loss  to  the  consumers  due  to  an  imperfect 
water  supply  which  had  an  esthetic  deficiency  of  100.  If  the  esthetic 
deficiency  were  less  than  100,  say  37,  then  the  loss  to  the  consumer 
would  be  yVt  of  $20,  or  $7 .  40  per  million  gallons.  In  other  words, 
the  figure  for  the  esthetic  deficiency  divided  by  5  gives  the  financial 
depreciation  value  of  the  water  supply  in  dollars  per  million  gallons, 

or// =  20 . 

100 


The  Value  of  Pure  Water  67 

Example:  Suppose  the  turbidity  of  a  water  is  3,  its  color  65,  and 
its  odor  2f  (that  is,  faintly  fishy),  because  of  the  presence  of  micro- 

I  2  -|-  '22  "i"  20 

scopic  organisms;  then  D  =  20 =$12.80;  that  is,  the  depre- 
ciation value  of  the  water,  because  of  its  unsatisfactory  physical 
qualities,  amounts  to  $12 .80  per  million  gallons. 

HARDNESS. 

The  point  at  which  a  water  becomes  objectionably  hard  has  never 
been  exactly  defined.  Standards  of  hardness  vary  in  different  parts 
of  the  country.  The  ordinary  person  washing  his  hands  considers 
the  water  soft  if  the  soap  will  quickly  produce  a  suds  without 
curdling.  A  hardness  of  10  parts  per  million  is  practically  unnotice- 
able,  and  it  requires  a  hardness  of  20  or  30  parts  per  million  to  produce 
''curdling."  Waters  which  have  a  hardness  below  25  parts  per  million 
seldom  cause  much  complaint,  but  when  the  hardness  rises  above  50 
the  water  is  well  entitled  to  the  appellation  "hard,"  and  above  100  it 
may  be  called  very  hard.  In  some  parts  of  the  country  hardnesses  of 
200  or  300  are  observed;   these  may  be  termed  "excessive." 

In  1903  a  number  of  experiments  were  made  by  the  writer  to  deter- 
mine the  effect  of  various  degrees  of  hardness  on  the  amount  of  soap 
used  in  washing  the  hands,  in  bathing,  and  in  general  household  uses. 
As  a  result  of  these  experiments  it  was  found  that  one  pound  of  the 
average  soap  as  used  in  the  household  would  soften  167  gallons  of 
water  which  had  a  hardness  of  20  parts  per  million.  This  was  equiva- 
lent to  about  three  tons  of  soap  per  million  gallons,  which  at  a  cost  of 
5  cents  per  pound,  would  amount  to  $300  per  million  gallons.  It  was 
found  also  that  for  every  increase  of  i  part  per  million  of  hardness  the 
cost  of  soap  increased  about  $10  per  million  gallons  of  water  softened. 

All  of  the  water  used  by  a  community  is  not  completely  softened. 
The  number  of  gallons  per  capita  per  day  completely  softened  has 
been  estimated  by  different  authorities  all  the  way  from  i  to  10.  It  will 
certainly  be  a  conservative  estimate  to  assume  that  one  gallon  per 
capita  is  thus   softened.     On  this  basis  the  depreciation  value  of 

water,  on  account  of  its  hardness,  is  D  =  —  ,     in    which  H  equals 

'  '  10  '  * 

the  hardness  of  the  water  in  parts  per  million,  and  D  the  depreciativc 
value  in  dollars  per  million  gallons. 


68  George  C.  Whipple 

Example :  Assume  the  total  hardness  of  a  water  to  be  79  parts  per 

79 
million ;  then  Z)  =  —  =  $7 .  90  per  million  gallons. 

This  takes  into  account  only  the  cost  of  soap  used  for  domestic 
purposes,  and  does  not  include  the  incidental  losses  and  inconveniences 
attendant  upon  the  use  of  hard  water  in  the  household.  These,  if 
they  could  be  expressed  in  terms  of  dollars  and  cents,  would  probably 
more  than  equal  the  cost  of  soap;  therefore  the  above  figures  err  on 
the  side  of  conservatism. 

TEMPERATURE. 

Everyone  knows  that  warm  water  is  unpalatable.  When  the  tem- 
perature rises  above  60°  F.,  people  do  not  like  to  drink  it  without 
cooling.  The  relation  between  the  temperature  of  the  water  and  the 
per  cent  of  objecting  consumers  may  be  represented  by  a  curve  based 

on  the   equation   />  = ,  in   which   p  equals  the  per  cent   of 

objecting  consumers,  and  d  equals  the  temperature  of  the  water  in 
Fahrenheit  degrees.  According  to  this  curve,  no  one  would  object 
to  drink  a  water  which  had  a  temperature  of  45°,  half  the  people 
would  object  at  66°,  and  all  would  object  at  75°.  If  it  is  assumed 
that  it  takes  one-half  pound  of  ice  per  capita  daily  to  cool  the 
water  used  for  drinking  during  four  months  in  the  year,  and  that 
ice  costs  30  cents  per  100  pounds,  then  the  depreciation  value  due 
to  temperature  would  be  equivalent  to  $5  per  million  gallons  of 
public  supply  for  100  per  cent  of  objecting  consumers,  assuming  the 

per  capita  consumption  to  be  100  gallons  daily,  otD  =  —  X  $5  =  ^^ — ^— ^ 

in  dollars  per  million  gallons,  in  which  d  =  the  average  temperature 
during  the  four  warmest  months  of  the  year.  This  may  be  considered 
as  the  depreciation  value  due  to  temperature.  The  temperature  of 
ground  waters  seldom  rises  above  60°  in  the  house  taps  even  in 
summer,  and  in  cities  supplied  with  ground  water  a  large  propor- 
tion of  the  consumers  do  not  use  ice.  Surface  waters,  on  the  other 
hand,  in  the  latitude  of  New  York,  generally  maintain  a  temperature 
of  60°  or  more  at  the  house  taps  for  at  least  four  months  of  the 
year.     The  temperature  factor  is  an  important  one  in  many  cases, 


The  Value  of  Pure  Water  69 

but  it  need  not  be  used  except  when  comparing  surface  waters  with 
ground  waters. 

In  a  similar  way  it  might  be  possible  to  calculate  the  reduced  value 
of  a  water  due  to  other  objectionable  characteristics,  such  as  the 
presence  of  large  amounts  of  iron  or  chlorine.  Except  in  special  cases, 
these  would  not  be  as  important  as  the  more  obvious  qualities  above 
described,  and  they  need  not  be  considered  in  this  discussion. 

SUMMARY  OF  PRINCIPAL  FORMULAE. 

Depreciation  due  to  sanitary  quality — 

I.     D=^2.ys(T-N). 

Depreciation  due  to  physical  characteristics — 

Pc  +  P^+Po 


2.      D  =  20- 


100 


c 

p,  =  Si^t 


po=20i+s.so:i+soi. 

Depreciation  due  to  hardness — 

^  10 

Depreciation  due  to  temperature — 

4.     D  =  — ,    m  which — 

180      ' 

D  =  the  depreciation  value  in  dollars  per  million  gallons; 

r  =  typhoid  fever  death-rate  per  100,000; 

iV  =  typhoid  fever  death-rate  assumed  to  be  due  to  causes 

other  than  water,  and  which  may  be  ordinarily  taken  as 

20  per  100,000; 
pc  =  peT  cent  of  consumers  who  object  to  the  color  of  the 

water; 
/>/  =  per  cent  of  consumers  who  object  to  the  turbidity  of  the 
water; 

Po  =  'peT  cent  of  consumers  who  object  to  the  odor  of  the  water; 
c  =  color  reading; 
/  =  turbidity  reading; 


70  George  C.  Whipple 

0^  =  odors  due  to  vegetable  matter,  expressed  according  to 
standard  numerical  scale; 

0(i  =  odors  due  to  decomposition,  expressed  according  to 
standard  numerical  scale; 

Oo  =  odors  due  to  microscopic  organisms,  expressed  accord- 
ing to  standard  numerical  scale; 

H  =  hardness  of  Water  in  parts  per  million ; 

d  =  average  temperature  of  water  during  four  warmest  months. 

APPLICATION  OF  THE  FORMULA. 

It  now  remains  to  apply  the  principles  above  set  forth  to  actual 
cases  and  see  to  what  conclusions  they  lead. 

effect  of  contamination. 

The  average  death-rate  from  typhoid  fever  in  American  cities 
which  have  more  than  30,000  inhabitants  is  about  35  per  100,000. 
Applying  formula  (i),  and  assuming  a  value  of  20  for  A^,  then 

^=2.75(35-2o)=$4i.25; 
that  is,  the  average  depreciation  value  of  the  water  supplies  of  our 
American  cities,  taken  as  a  whole,  is  $41 .  25  per  million  gallons  because 
of  their  unsanitary  quality,  or  about  $15,000  per  annum  for  each  mil- 
lion gallons  a  day  of  supply. 

The  above  figure  takes  into  account  both  good  and  bad  supplies. 
The  average  typhoid  fever  death-rate  in  those  cities  which  have  rea- 
sonably good  water  supplies  may  be  taken  in  round  numbers  as  about 
20,  while  in  those  cities  which  have  supplies  more  or  less  contaminated 
it  varies  from  this  up  to  40  or  60.  In  some  of  the  worst  cases  it  is 
more  than  100  per  100,000.  In  Pittsburg,  for  example,  the  typhoid 
death-rate  for  several  years  has  averaged  120.  Here,  according  to 
formula  (i),  D  =  2.75  (120— 20)  =$275  per  million  gallons.  This  is 
figured,  however,  on  a  per  capita  water  consumption  of  100  gallons 
a  day.  The  actual  consumption  is  about  250  gallons  per  capita  per 
day;  hence  D  should  be  taken  as  ^^^  of  $275,  or  $110  per  million 
gallons.  Each  million  gallons  of  polluted  Allegheny  River  water 
pumped  to  Pittsburg  has  therefore  reduced  the  vital  assets  of  the  com- 
munity by  $110.  This,  for  a  population  of  350,000,  amounts  to 
$3,850,000  per  year — a  sum  enormously  greater  than  the  cost  of 
making  the  water  pure. 


The  Value  of  Pure  Water 


71 


Classifying  water  supplies  according  to  their  source,  the  following 
will  give  a  general  idea  as  to  the  depreciation  value  of  various  types  of 
water  from  the  sanitary  standpoint,  based  on  general  average  typhoid 
fever  death-rates : 


Charactes  of  Water  Supply. 


Depreciation  Value  in 

Dollars    per    Million 

Gallons 


I. 


So. 00 

$0.00 

$  0.00  to  $  15.00 


Ground  waters,  except  in  cases  where  pollution  is  excessive,  or 
where  wells  are  driven  in  rock  or  soil  abounding  in  fissures     . 

2.  Filtered  waters  (assuming  modern  methods  of  construction 
and  operation), 

3.  Surface  waters — 

a)  Ordinar>'  upland  waters,  with  insignificant  contamination  . 

b)  Shghtly  contaminated  waters,  with  good  storage  in  lakes 
or  large  reservoirs 10.00    to      50.00 

c)  River  waters,  slightly  contaminated,  little  or  no  storage       .        25.00    to     100.00 

d)  River  waters,  much  contaminated,  little  or  no  storage     .     .        50.00    to    200.00 

EFFECT  OF  TURBIDITY,  COLOR,  AND  ODOR. 

It  has  been  shown  that  the  esthetic  deficiency  of  water  depends 
upon  three  variable  characteristics,  which  may  have  many  different 
combinations;    consequently,  it  is  difficult  to  classify  the  water  sup- 

TABLE  5. 
Examples  of  Waters  wrrH  Different  Physical  Characteri.stics. 


City 


Source  of  Supply 


Turbid- 
ity 


Color 


Odor 


Per  Cent 
of  Ob- 
jecting 
Con- 


Portland,  Me 

Boston,  Mass 

Cleveland,  Ohio.  . 
Worcester,  Mass . . 
New  York  City. .  . 
Brooklyn,  N.Y... 

Jersey  City,  N.  J.. 
Waterlown,  N.  V. 
Springfield,  Mass. 

Bangor,  Me 

Pittsburgh,  Pa.  . . 
Philadelphia,  Pa.. 
St.  Louis,  Mo. .  .  . 


Lake  Sebago 

Sudbury  and  Nashua  Rivers 

Lake  Erie 

Storage  Reservoirs 

Croton  River 

Ponds  and  driven  wells  on  Long 

Island 

Rockaway  River 

Black  River 

Ludlow  Reservoir 

Penobscot  River 

Allegheny  River 

Schuylkill  River 

Mississippi  River 


Depreci- 

ation\'al- 

ue  per 

Million 

Gals. 


ground  waters. 

Camden,  N.  J.. .  . 

Driven  wells 

0 

0 
0 

1 

0 

10 

0 
0 
0 

0 
0 

5 

0.00 

Flatbush,  L.  I. .  .  . 

Driven  wells 

o.oo 

Lowell,  Mass 

Driven  wells 

1 .00 

surface  waters. 

I 

IS 

2V 

20 

3 

2S 

2V 

30 

18 

5 

I.SV 

30 

2 

30 

y" 

40 

4 

20 

3" 

SS 

3 

13 

15^ 

^t 

4 

32 

2V  \g 

38 

6 

70 

3" 

SS 

S 

27 

AS 

104 

6 

6S 

T,v  im 

so 

64 

30 

iv  zm 

87 

ISO 

10 

3V  2m 

102 

200 

30 

3V2m 

127 

$  4.00 
6.00 
6.00 

8.00 
II  .00 

7.20 

7  60 
II  .00 
20.80 
11.80 
17.40 
20.40 
25.40 


Some  of  the  above  figures  do  not  represent  present  conditions.  For  example, 
Watertown,  N.  Y.,  now  has  filtered  water;  St.  Louis  uses  a  chemically  treated 
water;  etc. 


72 


George  C.  Whipple 


plies  of  the  country  on  this  basis.  For  this  reason  the  few  typical 
examples  given  in  Table  5  may  be  more  instructive  than  any  attempt 
at  a  general  classification. 

It  will  be  seen  from  the  above  figures  that,  while  the  general 
attractiveness  of  a  water  is  of  less  importance  than  its  sanitary  quality, 
yet  it  is  by  no  means  insignificant.  For  instance,  such  a  water  as 
that  now  supplied  to  New  York  City  from  the  Croton  River  has  a 
depreciation  value  of  $11  per  million  gallons,  or  nearly  a  million  and 
a  half  dollars  a  year  for  a  daily  supply  of  350  million  gallons.  At  4 
per  cent  this  represents  the  interest  on  about  $35,000,000,  a  sum  several 
times  as  large  as  the  cost  of  filtration.  An  algae-laden  water  like  that 
of  Ludlow  Reservoir  at  Springfield,  Mass.,  has  a  depreciation  value 
of  more  than  $20  per  million  gallons,  because  of  its  odor  and  turbidity. 
A  colored  water  like  that  of  the  Black  River  at  Watertown  before 
filtration  has  a  depreciation  value  of  $11,  while  a  turbid  water  like 
that  of  the  Mississippi  River  at  St.  Louis  gives  $25. 

In  most  surface  waters  the  physical  characteristics  vary  greatly  at 
different  times  of  the  year.  During  the  spring  and  fall,  for  instance, 
the  color  and  turbidities  may  be  high  on  account  of  rains,  while  during 
the  summer  the  water  may  have  bad  odors  due  to  microscopic  organ- 
isms. The  depreciation  value  of  a  certain  reservoir  water,  calculated 
as  above  described,  serves  well  to  show  this  seasonal  variation,  as 
illustrated  by  the  following  figures : 

TABLE  6. 

Seasonal  Variation  in  the  Depreciation  Value  of  a  Surface  Water  Due  to  Seasonal 
Changes  in  Turbidity,  Color,  and  Odor. 


Month 

January 

February 

March 

AprU 

May 

June 

July 

August 

September 

October 

November 

December 

Average 


Turbid- 

Color 

ity 

6 

25 

8 

28 

7 

27 

5 

22 

8 

25 

7 

30 

4 

22 

4 

25 

3 

30 

4 

28 

3 

26 

4 

25 

Odor 


(3^  o 
(3V  o 
(,3V  o 
(3V  2 

(3V  I 
(3V  1 
(3^  o 
(3^  o 


3v 
3v 
3v 
3v  + 

5    Org.  0.3m 
3m 

5    Org.  osm 
Org.  0.5OT 

Org.  0.5m 
Org.  o.sm 

3m 

3"« 


Per  Cent 
Objecting 
Consumers 


44 
47 
45 
40 

49 
48 
43 
62 

63 
49 
40 
42 


Depreciation  of 

Value  per 
Million  Gals. 


$  8.80 
9.40 

9.  GO 
8.00 

9.80 

9.60 

8.60 

12.40 

12  .60 
9.80 
8.00 
8.40 

$  9-53 


The  Value  of  Pure  Water 


73 


EFFECT    OF    HARDNESS. 

The  waters  of  New  England  are  comparatively  soft,  although  in 
some  instances  the  ground  waters  are  hard.  In  the  Middle  West,  on 
the  contrary,  most  of  the  surface  waters  are  quite  hard,  and  in  some 
cases  the  hardness  is  excessive.  The  following  figures  serve  to  give 
an  idea  of  the  range  in  the  depreciation  value  of  waters  due  to  hard- 
ness. 

TABLE  7. 


State 

City  or  Town 

Source  of  Supply 

Total 
Hardness 
(Parts  per 

Mill.) 

Deprecia- 
tion Value 
per  Million 
Gals. 

Maine 

Augusta 

WaterviUe 

Kennebec  River 

Messalonskee  River 

20 

15 
12 

33 

SO 

40 

64 

191 

179 

200 

335 
578 
215 
243 

S  2.00 
2  ^0 

Massachusetts  

Boston 

Sudbury  and  Na,shua  Rivers.  . 
Storage  Reservoir 

Cambridge 

3  30 
5  00 

A    00 

II 

Pittsfield 

Storage  Reservoir 

New  York 

New  York 

Croton  River 

Albany 

Hudson  River. 

6.40 
19     10 

«i 

Oswego 

Oswego  River 

Pennsylvania  

Philadelphia 

Schuylkill  River 

17  90 

Ohio 

Toledo 

Maumee  River 

Columbus 

Scioto  River 

33-50 

33-50 
21.52 
24  30 

1 

" 

Warren 

Mahoning  River      .  .    .  . 

England 

Chelsea  Company 

East  London  Company 

London  

EFFECT   OF   FILTRATION. 

Sanitary  quality. —  The  following  figures  show  to  what  extent  the 
sanitary  value  of  a  polluted  public  water  supply  is  increased  by  an 
efficient  system  of  filtration : 

Laurence,   Mass. — 

Water  supply,  Merrimack  River,  filtered  by  a  slow  sand  filter. 

Population  70,000. 

Water  consumption,  40  gallons  per  capita  daily. 

Before  filtration  the  typhoid  fever  death-rate  was  121   per   100,000;  since  then 
it  has  been   26. 

Before   filtration   2^  =  2.75    (121  — 20)  X  W  =$693. 

After  filtration   Z?  =  2.75    (26-20)  X  W  =$41- 

Increase  in  sanitary  value  =  $693  — $41  =$652   per  million  gallons,  or  $665,000 
per  year,  or  $9.50  per  year  per  capita. 
Albany,  N.  Y. — 

Water  supply,  Hudson  River,  filtered  by  sand  filter. 

Population,  95,000. 

Water  consumption,   165  gallons  per  capita  daily. 

Before  filtration  the  typhoid  fever  death-rate  was  104  per  100,000;  since  then 
it  has  been  26. 

Before  filtration  Z)  =  2.  75(104— 2o)X}§8  =$140. 

After    filtration    D  =  2.-j$    (26-20)  Xi?g  =  $10. 


74  George  C.  Whipple 

Increase  in  sanitary  value  =  $140— $io  =  $130  per  million  gallons,  or  $450,000 

per  year,  or  $4 .  75  per  capita  per  year. 
Binghamton,  N.   Y. — 

Water  supply,  Susquehanna  River,  filtered  by  a  mechanical  filter. 

Population,   42,000   (approximately). 

Water  consumption,   160  gallons  per  capita  daily. 

Typhoid  fever  death-rate  before  filtration,  49;  after  filtration,    11   per   100,000. 

Before   filtration  D  =  2.-js    (49- n)  Xifg  =$65. 

After  filtration  Z)  =  2.  75(11  — 11)    Xj-n  =  o. 

Increase  in  sanitary  value  =  $65.00  per  million  gallons,   or  $160,000  per  year, 

or  $3.80  per  capita  per  year. 
Watertown,  N.    Y. — 

Water  supply.   Black  River  filtered  by  mechanical  filter. 

Population,   25,500   (approximately). 

Water  consumption,    160  gallons  per  capita   daily. 

Typhoid  fever  death-rate    before    filtration,  68  per  100,000;  after  filtration,  19.5. 

Before  filtration  D  =  2.75   (68-20)  XTgu  =  $82. 50. 

After  filtration  ^  =  2.75   (20— 20)  X  VV  =o- 

Increase  in  sanitary  value  =  $82. 50  per  million  gallons,  or  $120,000  per  year, 

or  $4.75  per  capita  per  year. 

Illustrations  like  the  above  might  be  multiplied,  but  the  four  cases 
selected  are  sufficient  to  illustrate  the  general  fact.  It  is  easily  seen 
from  them  that  the  filtration  of  a  polluted  public  water  supply  increases 
to  a  very  great  extent  the  vital  assets  of  a  community,  and  the  increase 
in  most  cases  is  many  times  greater  than  the  cost  of  constructing  and 
operating  the  works.  Money  paid  to  the  doctor,  the  apothecary,  and 
the  undertaker  is  not,  in  one  sense,  a  loss  to  a  community,  as  it  is 
merely  a  transference  of  money  from  one  man's  pocket  to  another's, 
but  in  the  broader  sense  any  loss  of  productive  capacity  or  any 
unnecessary  expenditure  is  a  loss.  Deaths  from  typhoid  fever  and 
from  other  diseases,  however,  represent  a  very  material  loss  of  the 
productive  capacity  of  a  community,  and  consequently  a  decrease  in 
what  may  be  termed  the  "vital  assets."  In  the  case  of  the  city  of 
Albany,  for  instance,  the  increased  worth  of  the  water  to  the  city, 
because  of  its  efficient  filtration,  amounts  to  $475,000  per  year,  of 
which  at  least  $350,000  may  be  considered  as  a  real  increase  in 
the  vital  assets  of  the  city. 

If  in  the  formula  D=$2.js  (T-N)  we  let  T-N  =  i,  then  D  = 
$2.75;  that  is,  a  decrease  in  the  typhoid  fever  death-rate  of  i  per 
100,000  causes  an  increase  in  the  vital  assets  of  the  city  of  $2.75  for 
each  million  gallons  of  the  public  water  supply  (assuming  this  to  be 


The  Value  of  Pure  Water 


75 


loo  gallons  per  capita),  or  $o.io  per  capita  per  year  for  each  unit 
reduction  of  the  typhoid  fever  death-rate  per  100,000.  In  other  words 
the  decrease  in  the  typhoid  death-rate  per  100,000  divided  by  10  gives 
the  increased  vital  assets  of  the  community  in  dollars  per  capita  per 
year.  Thus  in  the  case  of  Albany,  above  given,  the  reduction  in  the 
typhoid  fever  death-rate  w^as  78  per  100,000.  On  the  basis  of  10  cents 
per  capita  per  unit  decrease,  this  would  amount  to  $0. 10  X  78X95,000 
=  $741,000  per  year,  assuming  a  per  capita  consumption  of  100  gallons 
daily,  or  $450,000  for  a  per  capita  consumption  of  165  gallons  daily, 
which  is  the  figure  stated  above. 

Looking  at  the  matter  in  another  way,  it  may  be  said  that  the  puri- 
fication of  a  polluted  water  is  a  sort  of  life-insurance  for  the  people, 
the  value  of  which  is  equal  to  10  cents  per  capita  for  each  unit  decrease 
in  the  typhoid  fever  death-rate  per  100,000  which  it  brings  about. 
Such  a  sum  capitalized  represents  a  large  amount  of  money.  In 
Albany,  for  example,  where  the  typhoid  fever  death-rate  has  been 
reduced  78  per  100,000,  the  annual  saving  of  life-value  would  be  $7 .  80 
per  capita.  Capitalized  on  the  basis  of  an  annual  life-insurance 
premium  of  $17  per  thousand,  this  would  represent  an  insurance 
policy  of  about  $460  per  year  for  each  inhabitant,  or  $2,300  for  each 
head  of  a  family. 

Physical  quality. — The  figures  of  Table  8  show  the  effect  of  filtra- 

TABLE  8. 


a 

3 

oi 

1 

-3 
•0  V 

4»  t; 

City 

Source  of 
Supply 

Type  of 
Filter 

Sample 

IS 

3 

8 

U 

Odor 

cU 

'C    4) 

HI 

Per  Mil 

J.  Gals. 

Lawrence,  Mass. 

Merrimack 

Slow  sand 

Raw 

10 

40 

3t  im 

S8 

$11.60 

River 

Filtered 

0 

40 

2v 

28 

S.6o 

$  6.00 

Albany,  N.  Y. 

Hudson  River 

Slow  sand 

Raw 

40 

32 

3v  im 

69 

1380 

Filtered 

2 

24 

2v 

27 

S.40 

8.40 

Yonkers,  N.  Y. 

Sawmill  Creek 

Slow  sand 

Raw 

6 

30 

^v  im 

49 

9.80 

Filtered 

0 

3 

iv 

4 

0.80 

9.00 

PouKhkeepsie, 

Hudson  River 

Slow  sand 

Raw 

30 

S5 

3v  im 

78 

17,60 

\.  Y. 

Filtered 

0 

30 

iK 

17 

3.40 

14.20 

Binghamton, 

Susquehanna 

Mechanical 

Raw 

30 

20 

3v 

S7 

II  .40 

N.  Y. 

River 

filter 

Filtered 

0 

S 

IV 

5 

1 .00 

10.40 

Watertown,  N.  Y. 

Black  River 

Mechanical 

Raw 

6 

70 

3v 

72 

14.40 

filter 

Filtered 

0 

8 

IV 

10 

a. 00 

12  .40 

LitdeFalU,  N.Y. 

Passaic  River 

Mechanical 

Raw 

20 

3S 

3V 

S6 

11.20 

filter 

Filtered 

0 

8 

IV 

10 

2.00 

9  20 

Brooklyn.  N.  Y. 

Baisley's  Pond 

Mechanical 

Raw 

15 

31 

av 

52 

10  40 

filter 

Filtered 

2 

3 

0 

7 

1 .40 

9  00 

76  George  C.  Whipple 

tion  on  the  attractiveness  of  waters — that  is,  upon  the  aggregate  effect 
of  their'physical  characteristics : 

The  above  figures  do  not  pretend  adequately  to  represent  the  con- 
ditions in  any  of  the  cities  included  in  the  list,  as  the  analysis  in  each 
case  represents  only  one  date.  They  are,  however,  typical  of  what 
the  filters  in  the  various  places  are  doing,  and  they  indicate  that  the 
increased  value  of  the  water,  because  of  its  filtration,  is  as  great  as 
the  cost  of  the  works — in  some  cases  it  is  even  greater.  Thus  if  the 
effect  of  filtration  on  the  sanitary  qualities  of  these  waters  is  entirely 
ignored'and  only  its  effect  on  their  physical  qualities  considered,  the 
filtration  of  these  supplies  would  still  be  a  profitable  undertaking  from 
a  financial  standpoint.  If  the  sanitary  qualities  were  also  considered, 
the  advantages  of  filtration  would  be  found  to  be  many  times  greater. 
This  phase  of  the  subject  has  not  received  the  consideration  it 
deserves,  and  it  is  this  topic  which  the  writer  desires  especially  to 
emphasize  in  the  present  paper. 

Water- softening. — The  following  figures  will  illustrate  the  financial 
value  of  water-softening  plants : 

Winnipeg,   Manitoba — 

Hardness  of  water  before  treatment 580 

Hardness  of  water  after  chemical  treatment  and  filtration 193 

Reduction  in  hardness 387 

Increased  value  of  water  due  to  water-softening  process,  per  million  gallons  $38.70 

Oberlin,  Ohio — 

Hardness  of  raw  water 1 70 

Hardness  of  raw  water  after  chemical  treatment  and  filtration 48 

Reduction  in  hardness 122 

Increased  value  of  water  due  to  water-softening  per  million  gallons   .     .     .  $12.20 

These  figures  refer  only  to  water  used  for  domestic  purposes.  If 
industrial  uses  also  were  considered  the  advantages  of  water  softening 
would  be  still  more  evident. 

At  the  present  time  there  are  not  many  water- softening  plants  in 
existence  in  connection  with  municipal  supplies,  but  the  advantages 
to  be  gained  are  very  great,  and  are  becoming  appreciated  by  the 
managers  of  railroads  and  industrial  establishments.  With  a  better 
understanding  of  the  practical  benefits  to  be  derived  from  the  use  of 
soft  water,  it  may  be  confidently  expected  that  during  the  next  10 
years  the  number  of  municipal  water-softening  plants  will  very  greatly 
increase. 


The  Value  of  Pure  Water  77 

SUMMARY. 

In  the  foregoing  paper  attention  has  been  called  to  the  following 
propositions : 

1.  Pure  water  as  compared  with  impure  water  has  a  real  financial 
value  to  a  community. 

2.  This  value  may  be  measured  by  determining  what  impure  water 
costs  the  community. 

3.  There  are  three  principal  characteristics  which  affect  the  value 
of  water  to  the  general  consumer — its  sanitary  quality,  its  general 
attractiveness,  and  its  hardness. 

4.  A  formula  is  suggested  for  computing  the  effect  of  the  sanitary 
quality  of  water  on  its  financial  value  to  a  community.  It  is  based  on 
the  typhoid  fever  death-rate. 

5.  A  formula  is  suggested  for  computing  the  effect  of  the  general 
attractiveness  of  water  on  its  value  to  consumers.  It  is  based  on  the 
physical  characteristics  of  turbidity,  color,  and  odor. 

6.  A  formula  is  suggested  for  computing  the  effect  of  the  hardness 
of  water  on  its  value  to  the  consumers.  It  is  based  on  the  use  of  soap 
in  the  household. 

7.  Considered  from  the  financial  aspect  alone,  and  disregarding  all 
humanitarian  considerations,  the  filtration  of  a  polluted  water  supply 
adds  very  greatly  to  the  vital  assets  of  a  community ;  hence,  as  a  mere 
business  proposition,  no  city  can  afiford  to  allow  an  impure  water  sup- 
ply to  be  publicly  distributed. 

8.  The  advantages  to  a  community  of  having  a  water  supply,  not 
only  safe,  but  also  attractive  in  appearance,  taste,  and  odor,  are 
material  from  a  financial  aspect.  The  increased  value  of  many  waters 
because  of  the  improvement  in  their  esthetic  qualities  alone  justifies 
the  cost  of  filtration. 

9.  Water-softening  at  present  does  not  receive  the  attention  it 
deserves  at  the  hands  of  municipal  authorities.  The  economic  advan- 
tages to  be  gained  by  removing  the  hardness  of  water  are  so  great 
that,  in  many  cases,  the  saving  to  the  ordinary  water-consumers  justi- 
fies the  cost  of  softening  water. 

10.  The  formulae  here  suggested  and  the  detailed  results  derived 
from  their  use  are  not  to  be  considered  as  of  great  accuracy,  as  the 
assumed  data  are  not  fully  adequate.       They  are  given  merely  to 


78 


George  C.  Whipple 


show  the  possibihty  of  computing  the  value  of  pure  water  in  terms  of 
dollars  and  cents,  and  to  illustrate  the  financial  value  of  filtration  and 
justify  its  cost. 

TABLE  0 

Depreciation  Due  to  SA^aTARY  Quality. 

Values  of  D  for  Different  Values  of  T-N  in  the  formula  D  =  2.ts  {T-N), 
Values  of  D  in  Dollars  for  Million  Gallons. 


T-N 

0 

I 

2 

3 

4 

5 

6 

7 

8 

9 

o. . 

0.00 

2.75 

5-50 

8.25 

II  .00 

13-75 

16.50 

19  25 

22.00 

24-75 

lO.  . 

27.50 

30.25 

33  00 

35-75 

38.50 

41-^5 

44.00 

46.75 

49-50 

52-25 

20.  . 

5500 

57-75 

60.50 

63-25 

66.00 

68.75 

71-50 

74-25 

77-00 

79-75 

30.. 

82.50 

85-25 

88-00 

90.75 

93  50 

96.25 

99.00 

101-75 

104.50 

107-25 

40.. 

no.  00 

112.75 

115-50 

118.25 

121 .00 

123-75 

126.50 

129.25 

132.00 

134-75 

50.. 

137-50 

140-25 

143-00 

145-75 

148-50 

151-25 

154.00 

156.75 

159-50 

162.25 

60.. 

165.00 

167.55 

170.50 

173-25 

I  76 . 00 

178-75 

181.50 

184-25 

187.00 

189-75 

70.. 

192  so 

195-25 

198.00 

200.75 

203.50 

216.25 

209.00 

211.75 

214.50 

217.25 

80.. 

220.00 

222.75 

225.50 

228.25 

231.00 

233-75 

236.50 

239-25 

242.00 

244-75 

90.  . 

247-50 

250.25 

253-00 

255-75 

258.50 

261.25 

264.00 

266.75 

269.50 

272.25 

100. . 

275.00 

277-75 

280.50 

283-25 

286.00 

288.7s 

291-50 

294-25 

297.00 

299-75 

110.  . 

302 . 50 

305-25 

308.00 

310.75 

313-50 

316.25 

319.00 

321-75 

324-50 

327-25 

120.  . 

330.00 

332.75 

335-50 

338.25 

341 -00 

343-75 

346  -  50 

349-25 

352  -  00 

354-75 

130.  . 

357-50 

360.25 

363 ■ 00 

365-75 

368.50 

371-25 

374-00 

376.75 

379-50 

382.25 

140. . 

385.00 

387-75 

390-50 

393  25 

396.00 

398  -  75 

401  -  50 

404 -25 

407 . 00 

409  -  75 

150.. 

412.50 

415-25 

418.00 

420-75 

423-50 

426.25 

429.00 

431  -  75 

434-50 

437-25 

TABLE  10. 

Esthetic  Deficiency  Due  to  Turbidity. 

Values  of  P^  for  Different  Values  of  t  in  the  Formula  ^,  =  5V  /. 
Per  Cent  of  Objecting  Consumers. 


Tur- 

bidity 

0 

I 

2 

3 

4 

5 

6 

7 

8 

9 

0  - .  -  - 

5-00 

7  OS 

8.66 

10.00 

11-15 

12-20 

13.20 

14. 10 

15.00 

10  ... . 

15.80 

16-55 

17 

30 

18.00 

18.70 

19 

35 

20.00 

20.60 

21  -20 

21.75 

20  ... . 

22.35 

22.91 

23 

45 

23  95 

24-45 

25 

00 

2545 

25.95 

26.45 

26.90 

30  ... . 

27-35 

27-80 

28 

25 

28.70 

29.15 

29 

55 

30.00 

30.40 

30.80 

30.90 

40 

31-60 

32-00 

32 

40 

32.75 

33.15 

33 

50 

33  90 

34.25 

34-60 

35.00 

50 

35-35 

35-70 

36 

05 

36.40 

36.70 

37 

OS 

37.40 

37.70 

38-05 

38.40 

60  . .  -  - 

38-70 

39-05 

39 

35 

39  65 

40.00 

40 

30 

40.60 

40.90 

41 .20 

41.50 

70 

41-83 

42-13 

42 

42 

42.72 

43  00 

43 

30 

43.58 

43.87 

44.15 

44.44 

80  ... . 

44.72 

45-00 

45 

27 

45-55 

45-82 

46 

09 

46.35 

46-73 

46.80 

47.16 

90 

47-43 

47.69 

47 

95 

48.21 

48.47 

48 

73 

48.98 

49-24 

49-49 

49.74 

100  . . . 

50-00 

Tur- 
bidity 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

TOO  .  .  . 

50-00 

52.44 

54-77 

57-00 

59.16 

61.23 

63  24 

65.19 

67.08 

68.92 

200  .  .  . 

70.71 

72-45 

74-16 

75.82 

77-45 

79  05 

80.62 

82.15 

83.66 

85.16 

300  .  .  . 

86.60 

88.00 

89.44 

90.83 

92.19 

93.54 

94.86 

96.17 

97.46 

98.74 

400  ..  . 

100.00 

lOI .22 

102.46 

103.68 

104.88 

106.16 

107.23 

108.39 

109.54 

110.67 

500  .  .. 

I 1 I . 80 

112. 91 

114.01 

115.10 

116. 18 

117.26 

118.32 

119.37 

120.40 

121.44 

600  .  .  . 

122.47 

123-49 

124.49 

125. 49 

126.49 

127.47 

128.45 

129.42 

130.38 

131.33 

700  .  .  . 

132.27 

133-22 

134.16 

135.09 

136.01 

136.93 

137.84 

138.74 

139  64 

140-53 

800  -  .. 

141.42 

142-21 

143.17 

144 .  04 

144.91 

145.77 

146.62 

147.47 

1 48 . 1 2 

149- 16 

900  .  .  . 

150.00 

150-83 

151-65 

152. 47 

153.49 

154.11 

154.91 

155.72 

156.52 

157-32 

1,000.  . 

158.11 

The  Value  of  Pure  Water 


79 


TABLE  II. 

Esthetic  Dkficiency  Due  to  Color. 

c 
Values  of  p^  for  Different  Values  of  c  in  the  Formula  ^^  =  — 

Per  Cent  of  Objecting  Consumers. 


Color 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

o  .  .  .  . 

OS 

1 .0 

1.5 

2  .0 

25 

3.0 

3-5 

4.0 

45 

lO   .  .  .  . 

50 

5-5 

6.0 

6.5 

70 

7.5 

8.0 

8.5 

9.0 

9   5 

20  ... . 

10.0 

10.5 

II  .0 

11.5 

12.0 

12.5 

130 

13-5 

14.0 

14-5 

30  — 

150 

15.5 

16.0 

16. s 

17.0 

17.5 

18.0 

18.5 

19.0 

19.5 

40  — 

20.0 

20.  s 

21.0 

21. S 

22.0 

22. s 

23.0 

23  5 

24.0 

24.5 

50  ... . 

25.0 

25-5 

26.0 

26.5 

27.0 

27-5 

28.0 

28.5 

29.0 

29. 5 

60  ... . 

30.0 

30.  5 

310 

31. s 

32.0 

32    5 

33   0 

33.5 

34.0 

34.5 

70  ... . 

35.0 

35-5 

36.0 

36.5 

37.0 

37.5 

38.0 

38.5 

39.0 

39.5 

80  ... . 

40.0 

40.5 

41 .0 

41. 5 

42.0 

42.5 

43   0 

43-5 

44.0 

44-5 

go 

45.0 

45-5 

46.0 

46.5 

47.0 

47.5 

48.0 

48.5 

49.0 

49.  5 

loo  ... 

50.0 

50.5 

51  0 

51-5 

52. 0 

52.5 

53   0 

53   S 

54.0 

54-5 

110  .  . . 

55. 0 

55.5 

56.0 

56.5 

57. 0 

575 

58.0 

58.5 

590 

59.  5 

120  .  . . 

60.0 

60.5 

61 .0 

61.5 

62.0 

62.5 

63.0 

63    5 

64.0 

64.5 

130  ..  . 

65  .0 

65.5 

66.0 

66. s 

67.0 

675 

68.0 

68.5 

69.0 

69.5 

140  .  .  . 

70,  0 

70.5 

71.0 

71    5 

72.0 

72.5 

73  0 

73.5 

74.0 

74.5 

150  .  . 

75-0 

75   5 

76.0 

76. 5 

77.0 

77.5 

78.0 

78.5 

79.0 

79-5 

160  .  .  . 

80.0 

80.5 

81.0 

81.5 

82.0 

82. 5 

83.0 

83.  5 

84.0 

84.5 

170  ..  . 

85.0 

85.5 

86.0 

86.  s 

87.0 

87. S 

88.0 

88.5 

89.0 

89.5 

180  ..  . 

goo 

go. 5 

91 .0 

91. S 

92.0 

92.5 

93.0 

93.5 

94.0 

94-5 

190  ..  . 

95  0 

95.5 

96.0 

96.5 

97.0 

97.5 

98.0 

98.  5 

99.0 

90.5 

200  .  .  . 

100. 0 

TABLE  12. 

Esthetic  Deficiency  Due  to  Odor. 

Values  of  />„  for  Diflerent  Values  of  O^  ,0^,  and  O^  in  the  Formula  /><,  =  205,  +  3. 50^+5  O'^. 

Per  Cent  of  Objecting  Consumers. 


Odor 

Vegetable  Odor 
(O  ) 

Odor  of  Decom- 
position 

Odor  due  to 
Organisms 

None 

Very  faint 

Faint 

Distinct 

Decided 

Strong 

0.0 
2.0 
8.0 

18.0 
32.0 
50.0 

0.0 
3.5 
14.0 
31.5 
56.0 
87.5 

0.0 

I              

so 

20.  J 

45.0 

80.0 

r                   

125.0 

TABLE  13. 
Depreciation  Due  to  Hardness. 
Values  of  D  for  Different  Values  of  H  in  the  Formula  D  = 
Depreciation  in  Dollars  per  Million  Gallons. 


n 


Hard- 
ness 

0 

I 

2 

3 

4 

5 

6 

7 

8 

9 

0 

10 

20 

30 

40 

50 

60 

lo::::: 
90 

1. 00 
2.00 
3.00 
4.00 

5.00 
6.00 
7.00 
8.00 
9.00 

0. 10 
1. 10 
2. 10 
3.10 
4.10 

5.IO 
6. 10 
7.10 
8.10 
9.10 

0.20 
1.20 
2.20 
3.20 
4.20 

5   20 
6.  20 
7.20 
8.20 
9.  20 

0.30  ; 
1.30 
2.30 
330 
4- 30 

5.30 
6.30 
7.30 
8.30 
930 

0.40 
1.40 
2.40 
340 
4.40 

5.40 
6.40 
7.40 
8.40 
9.40 

o.so 
I   50 
2.50 
350 
4.50 

5.50 
6.  so 
7SO 
8.50 
9.50 

0.60 
1.60 
2.60 
3.60 
4.60 

5. 60 
6.60 
7.60 
8.60 
9.60 

0.70 
1.70 
2.70 
3.70 
4.70 

5- 70 
6.70 
7.70 
8.70 
9.70 

0.80 
1.80 
2.80 
380 
4  80 

5. 80 
6.80 
7.80 
8.80 
9.80 

0.90 
1.90 
2.90 
3   90 
4.90 

S-90 
6.90 
7.90 
8.90 
9.90 

8o 


George  C.  Whipple 


TABLE    13— Continued. 


Hard- 
ness 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100.. . . 

10.00 

11.00 

12.00 

13  00 

14.00 

I?.  00 

16.00 

17.00 

18.00 

19.00 

200.. . . 

20.00 

21.00 

22.00 

23.09 

24.00 

25   00 

26.00 

27.00 

28.00 

29.00 

300.... 

30.00 

3100 

32.00 

33  00 

3400 

35   00 

36.00 

37.00 

38.00 

39.00 

400. .  . . 

40.00 

41 .00 

42.00 

43  00 

44.00 

45.00 

46.00 

47.00 

48.00 

49  00 

Soo.... 

50.00 

51.00 

52.00 

S3  00 

54- 00 

55  00 

56.00 

57.00 

58.00 

59.00 

600. . . . 

60.00 

61 .00 

62.00 

63.00 

64.00 

65.00 

66.00 

67.00 

68.00 

69.00 

700. .  .  . 

70.00 

71.00 

72.00 

73.00 

74.00 

75.00 

76.00 

77.00 

78.00 

79.00 

800..  .  . 

80.00 

81 .  00 

82.00 

83.00 

84.00 

85.00 

86.00 

87.00 

88.00 

89.00 

900. . .  . 

90  00 

91.00 

92.00 

93   00 

94  00 

95- 00 

96.00 

97   00 

98.00 

99.00 

1,000.  . 

100.00 

TABLE  14. 
Depreciation  Due  to  Temperature. 

Values  of  D  for  Different  Values  of  d  in  the  Equation  D  —  — r —  . 

180 
Dollars  per  Million  Gallons. 


Tem- 

pera- 

0 

I 

2 

3 

4 

5 

6 

7 

8 

9 

ture 

40 

0.022 

0.005 

0.089 

SO 

OI34 

0.200 

0.272 

0-355 

0.450 

0.56 

0.68 

0.80 

0.94 

1.09 

60 

0.2s 

I-2S 

1. 61 

1.80 

2.01 

2.22 

2-45 

2.69 

2.94 

3   20 

70 

3-45 

3-75 

4.05 

4.35 

4.68 

5.00 

5-34 

5  69 

6.05 

6.43 

80 

6.81 

7.20 

7.60 

8.03 

8.45 

8.90 

0-35 

9.80 

10.28 

10.  75 

A  CONTRIBUTION  TO  THE  GENERAL  PRINCIPLES  OF 
THE  PHARMACODYNAMICS  OF  SALTS  AND  DRUGS.* 

A.  P.  Mathews. 

{From  the  Laboratory  of  Physiological  Chemistry  of  the  University  of  Chicago.) 

This  paper  is  a  continuation  of  those  already  published/  which 
have  had  for  their  object  the  investigation  of  the  means  by  which  saUs 
and  drugs  influence  the  processes  going  on  in  hving  matter,  and  thus 
produce  the  phenomena  of  stimulation  and  depression. 

PART    I.     PHARMACODYNAMIC    ACTION    DUE    TO    IONS. 

The  cause  of  the  pharmacological  action  of  salts  upon  protoplasm 
has  been  the  subject  of  numerous  investigations,  but  until  the  develop- 
ment of  the  ionic  theory  these  investigations  had  led  to  no  further 
result  than  to  show  that  in  groups  of  similar  metals  the  heavier  were 
frequently  the  more  poisonous.  The  application  of  the  ionic  theory 
first  brought  some  order  into  this  part  of  pharmacology.  The  work 
of  the  American  investigators  Kahlenberg  and  True*  and  Heald,  con- 
firmed as  it  has  been  by  Kronig  and  Paul,  Hober,  True,  and  many 
others,  has  shown  in  the  clearest  manner  that  there  is  a  close  paral- 
lehsm  between  toxicity  and  the  state  of  ionization  of  many  of  the 
metals,  so  that  these  authors  conclude  that  the  pharmacological  action 
of  any  salt  solution  is  a  function,  in  large  measure  at  least,  of  the  ions 
into  which  the  salt  dissociates. 

This  general  conclusion  is,  in  my  opinion,  as  firmly  established  as 
is  the  conclusion  that  the  chemical  reactions  of  such  solutions  are  due 
to  the  ions  they  contain.  Indeed,  the  conclusion  is  a  necessary  result 
of  the  ionic  theory,  since  the  chemical  reactions  in  protoplasm  do  not 
differ  in  nature  from  those  going  on  elsewhere ;  and  if  salts  enter  into 
other  chemical  reactions  by  their  ions,  they  probably  enter  also  in  the 
same  manner  into  the  reactions  of  protoplasm. 

But  while  this  general  theory  is  a  great  step  forward,  it  stumbles 
against  the  objection  that  many  compounds  profoundly  affect  proto- 

*  Received  for  publication  March  23,  1906. 

»  \Lathews,  Amer.  Jour.  Physiol.,   1904,   10,  p.  291;   1904,   11,  p.  455;  1905,  14,  p.  304;  1905,   12, 

p.  421;    1904,    II,    p.    2j8. 

■  Kahlenberg  and  True,  Botariical  Gazette,  1896,  22,  p.  91. 

81 


82  A.  P.  Mathews 

plasm,  although  they  do  not  dissociate  electrolytically  into  ions  in  the 
ordinary  sense  of  the  term.  To  explain  the  action  of  such  compounds 
as  ether  and  organic  drugs,  either  one  must  fall  back  upon  the  assump- 
tion of  the  action  of  undissociated  molecules,  or  the  idea  of  dissociation 
must  be  extended  to  cover  dissociation  which  is  not  accompanied  by 
electrical  conductivity.  Kahlenberg  and  True,  and  indeed  nearly  all 
observers,  have  adopted  the  theory  that  some  action  must  be  ascribed 
to  undissociated  molecules;  but  it  appears  to  me,  in  view  of  the  fact 
that  such  another  kind  of  dissociation  is  well  known  to  occur — as,  for 
example,  the  dissociation  of  NH^OH  into  NH3  and  H^O — and  also 
that  this  dissociation  has  been  shown  by  Nef '  to  determine  the  chemi- 
cal reactions  of  such  compounds,  that  the  alternative  of  the  action  of 
dissociated  particles  is  the  more  probable.  At  any  rate,  it  would  be 
premature  to  ascribe  pharmacological  action  to  undissociated  mole- 
cules until  the  possibilities  that  that  action  is  due  to  the  dissociated 
particles  shall  have  been  proved  to  be  insufificient.  In  the  present 
paper  I  shall  deal  with  pharmacological  action  due  to  particles  dis- 
sociated as  ions,  and  in  a  subsequent  paper  to  action  due  to  non-ionic 
dissociation.  The  general  principles  which  I  have  worked  out  apply 
primarily  to  ionic  particles,  but  I  think  it  altogether  probable  that 
they  will  be  found  to  apply  equally  well  to  non-ionic  dissociation, 
since  there  is  in  all  likelihood  no  essential  difference  in  kind  between 
such  dissociation  as  that  of  NH^OH  into  NH3  and  H^O  and  ionic 
dissociation.  The  two  probably  differ  only  in  that  in  the  one  case 
the  two  electrical  charges  are  on  the  same  particle,  whereas  in  ionic 
dissociation  they  are  on  separate  atoms.* 

While,  then,  it  cannot  be  denied  that  some  action  may  be  referable 
to  undissociated  molecules,  the  clear  parallehsm  between  dissociation 
and  pharmacological  action  in  the  case  of  salts,  and  the  equally  clear 
parallelism  between  non-ionic  dissociation  and  pharmacological  action 
in  organic  compounds,  indicates  to  my  mind  that  it  is  to  these  disso- 
ciated particles  as  the  possible  cause  of  pharmacodynamic  action  that 
attention  should  first  be  directed. 

Assuming,  therefore,  that  the  action  of  salts  is  due  in  chief  meas- 
ure to  the  ions  of  the  solution,  the  first  question  to  be  answered  is: 

'  Nef,  Liebig's  Annalen,  1904,  335,  p.  192. 

'  Mathews,  Biological  Bulletin,  1905,  8,  p.  342.     See  also  Nernst,  Theoretiscke  Ckemie,  4th  ed., 
1903.  P-  378. 


Pharmacodynamics  of  Salts  and  Drugs  83 

What  enables  any  ion  to  act  at  all  ?  What  makes  a  mercury  ion, 
for  example,  so  enormously  more  toxic  than  a  calcium  or  magnesium 
ion  ?  The  answer  to  this  question,  as  I  shall  now  proceed  to  show, 
is,  that  the  mercury  ion  has  an  enormously  greater  ionic  potential  than 
the  calcium  ion. 

IONIC  POTENTIAL   AND   PHYSIOLOGICAL   ACTION. 

In  an  earlier  paper'  the  term  "ionic  potential"  was  suggested  to 
designate  the  tendency  (>f  any  ion  or  atom  to  change  its  electrical  state. 
Bodlander  has  used  the  term  "  Haftintensitat "  to  designate  the  same 
factor,  and  he  and  Abegg  have  presented  evidence  to  show  that  the  ionic 
potential  is  one  of  the  chief  factors  in  determining  chemical  affinity. 

The  idea  that  this  property  of  the  ions  of  the  salt  might  be  of 
importance  in  determining  their  physiological  action  was  first  suggested 
by  my  colleague,  Dr.  J.  Stieglitz,  at  the  meeting  of  the  American 
Physiological  Society  in  Chicago  in  December,  1901.  At  that  time 
the  importance  and  real  bearing  of  the  suggestion  were  not  appre- 
ciated by  me,  but  about  a  year  or  so  later  I  was  much  struck  by  the 
fact  that  the  arrangement  of  the  metals  according  to  their  solution 
tensions,  as  given  by  Nernst,  was  practically  the  same  as  an  arrange- 
ment in  the  order  of  their  toxic  actions.  Stieglitz's  suggestion 
appeared  to  me  in  a  new  light,  and  I  set  to  work  to  get  additional 
evidence  that  it  is  the  ionic  potential  which  chiefly  determines  the 
physiological  action  of  ions.  In  1904  I  published  results  showing 
the  remarkable  parallelism  between  toxicity  and  ionic  potential  in  the 
action  of  salts  on  the  eggs  of  Fundulus  heteroclitus.  In  that  paper 
I  showed  that  valence  and  ionic  velocity — factors  to  which  main 
importance  had  been  attached  by  Hardy,  Loeb,  Pauli,  Posternak,  and 
which  have  been  recently  emphasized  by  Robertson* — are  unim- 
portant when  compared  with  the  importance  of  the  ionic  potential  as 
a  determining  factor  of  toxicity.  I  showed  also  that  the  phenomena 
of  stimulation  of  the  motor  nerve  by  salts  demonstrate  the  same  rela- 
tionship over  again,  and  in  the  clearest  and  most  decisive  manner. 
Inasmuch  as  I  had  already  interpreted  the  phenomena  of  chemical 
stimulation  of  motor  nerves  to  mean  that  the  nerve  impulse  was  due 
to  a  progressive  coagulation  of  the  colloids  of  the  nerve,  it  was  a 

■  Mathews,  Amer.  Jour.  Physiol.,  1904,  11,  p.  456. 

'  Robertson,  Trans.  Roy.  Soc.  oj  South  Australia,  1905,  29,  p.  11. 


84  A.  P.  Mathews 

necessary  inference,  if  this  were  true,  that  the  ionic  potential  must  be 
of  decisive  value  in  determining  the  precipitation  of  colloids  by  electro- 
lytes. An  investigation  of  this  possibility  showed  that  this  was 
indeed  the  case.  McGuigan'  then  investigated  the  relation  between 
the  ionic  potential  and  the  power  of  salts  to  prevent  the  action  of  the 
diastatic  ferment  upon  starch,  and  found  here  also  a  remarkably  close 
agreement  with  the  theoretical  anticipations. 

The  theory  of  the  importance  of  the  ionic  potential  has,  therefore, 
been  abundantly  confirmed.  It  is  the  more  surprising  that  it  has 
met  with  little  acceptance  or  attracted  little  notice,  since  its  general 
bearings  are  exceedingly  important,  involving  as  they  do  the  nature 
of  chemical  affinity  on  the  one  hand,  and  the  basis  of  pharmacology 
on  the  other. 

Owing  to  the  importance  of  the  subject,  the  slight  attention  it  has 
received,  and  to  the  fact  that  my  own  ideas  have  become  during  the 
course  of  the  work  more  clear  and  definite,  it  seemed  to  me  desirable 
that  the  results  previously  presented  both  by  myself  and  by  others  be 
summarized  and  put  in  a  more  definite,  and  perhaps  a  more  com- 
prehensible, form,  together  with  new  observations  in  the  same  direc- 
tion. Since  the  solution  tension  and  ionic  potential  are  properties 
with  which  physiologists  are  not  generally  very  familiar,  since  they 
lie  in  another  field  not  hitherto  brought  into  relationship  with  physio- 
logical processes,  I  have  tried  to  get  these  ideas  clear  at  the  outset. 

a)  General  physical  principles  involved  in  chemical  stimulation  and 
toxicity. — Any  physiological  response  to  an  external  agent,  however 
that  response  is  produced,  implies  a  change  in  motion  or  in  state  of 
the  atoms,  molecules,  and  masses  composing  the  protoplasmic  system. 
Now  such  a  change  in  state  means  that  work  has  been  done  in  pro- 
ducing these  movements,  and  this  work  must  have  been  done  at  the 
expense  either  of  the  internal  energy  of  the  system  itself,  or  of  the 
energy  of  the  environment — in  this  case  of  the  substance  causing  the 
change.  There  are  accordingly  two  possible  ways  in  which  an  exter- 
nal agent  such  as  a  salt  might  produce  a  change  in  the  protoplasmic 
system.  It  may  itself  supply  the  energy,  in  whole  or  in  part,  which 
is  necessary  to  bring  to  pass  the  internal  movements  of  the  system; 
or  it  may  by  its  presence  facilitate  the  transference  of  the  potential 

■  McGuiGAN,  Amer.  Jour.  Physiol.,  1904,  10,  p.  444. 


Pharmacodynamics  of  Salts  and  Drugs  85 

energy  of  the  system  itself  into  kinetic.  The  first  method  of  action  is 
clear,  but  a  word  may  be  said  as  regards  the  second.  Protoplasm, 
both  in  its  chemical  and  physical  aspects,  shows  many  of  the  phenom- 
ena of  false  equilibrium.  It  is  as  if  there  were  considerable  differ- 
ences of  potential  in  the  protoplasm  itself,  but  these  differences  were 
unable  to  neutralize  or  equalize  themselves,  owing  to  the  presence  of 
certain  resistances.  It  is  conceivable  that  our  ions  may  produce  results 
simply  by  acting  as  conductors,  or  in  removing  resistances;  acting,  in 
other  words,  as  catalytic  agents,  without  specifying  more  in  detail 
exactly  how  these  act.  As  an  example  of  this  kind  of  an  action  I  may 
mention  the  generation  of  the  nerve  impulse  when  a  motor  nerve  is 
suddenly  immersed  in  a  salt  solution,  or  when  its  cut  and  longitudinal 
surfaces  are  connected  by  a  wire.  In  this  case  the  wire  or  the  elec- 
trolyte serves  by  its  presence  only  to  equalize  the  difference  in  poten- 
tial between  the  two  surfaces,  and  the  nerve  stimulates  itself  by  its 
own  energy.  And  any  electrolyte  or  any  conductor  will  accomplish 
this  result.  To  what  extent  electrolytes  may  thus  affect  protoplasmic 
motions  cannot  be  foretold,  but  it  is  certainly  possible,  and  I  think  on 
the  whole  probable,  that  some  of  the  actions  of  salts  will  be  found  to 
be  of  this  nature.  In  such  cases  the  energy  content  of  the  salt  would 
be  of  little  importance.  But  while  it  cannot  be  denied  that  some  of 
the  salt  action  may  be  of  this  character,  few  specific  instances  are 
known  to  me. 

In  the  second  place,  salts  may  appear  to  act  catalytically  by  means 
of  their  valence  by  bringing  about  combinations  between  two  sub- 
stances, this  combination  resulting  in  one  substance  hastening  the 
decomposition  of  the  other.  The  ferments,  for  example,  may  in  this 
way  be  mordanted,  as  it  were,  by  some  bivalent  ions  to  the  substances 
they  ferment,  in  the  manner  suggested  by  Henri.'  It  will,  however, 
be  apparent  in  this  case  that  the  power  of  the  ion  to  form  such  com- 
binations of  the  right  degree  of  looseness  from  which  the  ferment  can 
again  escape,  must  be  dependent  on  the  chemical  aflfinity  of  the  ion. 
Since  the  chemical  affinity  is  very  probably  a  function  of  the  ionic 
potential,  this  case  also  really  brings  us  back  to  the  ionic  potential  as 
a  highly  important  factor  in  the  ion's  action. 

We  may  now  turn  from  these  hypothetical  cases  to  the  other  possi- 

»  Henri,  Revue  geiUrale  des  sciences,  1905,  16th  year,  p.  641. 


86  A.  P.  Mathews 

bility  in  which  saUs  affect  the  protoplasmic  movements  in  virtue  of 
their  own  energy  content. 

In  this  case  also  the  action  of  the  salt  may  be  twofold.  It  may 
either  change  the  whole  protoplasmic  system  by  means  of  the  energy 
in  the  salt,  or  it  may  by  a  transfer  of  a  portion  of  its  energy  to  one  part 
of  the  protoplasm  produce  such  a  change  in  the  latter  that  energy  is 
set  free  by  the  protoplasm  itself.  It  is  clear,  in  other  words,  that  the 
salt  may  destroy  the  protoplasm  either  directly,  in  virtue  of  a  great 
interchange  of  energy  between  itself  and  the  protoplasm,  or  it  may 
destroy  it  indirectly,  by  acting  on  some  part  of  the  protoplasm  in 
such  a  way  that  its  own  energy  destroys  it,  or  that  the  normal  con- 
version of  potential  into  kinetic  energy  necessary  for  the  continuance 
of  the  vital  processes  is  checked. 

A  distinction  is  generally  made  between  these  two  forms  of  destruc- 
tion, in  that  substances  acting  in  the  first  manner  are  said  to  be  imme- 
diately fatal;  those  acting  in  the  second  manner  are  said  to  exhaust 
the  protoplasm  by  over-stimulation  or  depression.  Thus  mercuric 
chloride  in  large  doses  probably  produces  an  immediate  coagulation 
and  destruction  of  the  living  matter.  In  this  case  an  immediate  and 
complete  change  in  the  protoplasmic  system  would  be  produced  by 
the  transfer  of  energy  from  the  salt  to  the  protoplasm  as  a  whole.  On 
the  other  hand,  mercuric  chloride  may  destroy  living  matter  in  small 
doses,  not  by  this  method,  but  by  bringing  about  a  small  change  in 
the  protoplasm,  by  means  of  which  internal  resistance  of  some  kind 
is  withdrawn  or  increased,  and  the  protoplasm  destroys  itself.  In 
both  these  cases,  however,  the  destruction  of  the  protoplasm  is  a 
direct  result  of  the  energy  content  of  the  salt,  and  salts  will  be  poison- 
ous according  as  the  amount  of  free  energy  in  them  is  great  or  small. 

For  all  salts  and  compounds  producing  changes  in  the  proto- 
plasmic system  in  the  two  last  ways  the  chemical  composition  will 
be  of  little  or  no  importance ;  the  sole  or  most  important  factor  deter- 
mining action  will  be  the  potential  and  amount  of  the  energy  in  it. 
The  character  of  the  carrier  of  the  energy,  in  other  words,  is  imma- 
terial. 

The  foregoing  considerations  may  be  expressed  in  a  formula  : 

Poisonous  action  of  any  sali—work  done  by  it  =  avail- 
able energy  in  it = amount  of  energy  X  its  potential. 


Pharmacodynamics  of  Salts  and  Drugs  87 

In  the  action  of  salts  on  protoplasm  we  have  to  deal,  then,  with  a 
transfer  of  energy  from  the  ions  to  the  protoplasm,  or  vice  versa. 
From  the  general  principles  of  physics  we  conclude  that  the  physiolo- 
gical action  of  any  salt  solution  must  be  a  function  of  its  energy  content. 
The  question  arises  how  this  energy  content  is  to  be  measured. 

It  has  been  shown  that  much,  if  not  all,  of  the  action  of  salt  solu- 
tions is  due  to  the  ions  present.  We  must,  therefore,  measure  the 
energy  content  of  the  ions.  The  total  energy  of  the  ion  is  composed 
of  two  factors,  the  free  or  available  energy  and  the  bound  energy.  It 
is  only  the  free  energy,  or  that  which  can  be  transferred  to  or  from 
the  ion,  which  is  of  importance  in  this  connection. 

1.  The  potential  factor  0}  the  free  energy. — The  interchange  of 
energy  between  the  salt  solution  and  the  protoplasm  must  depend 
on  the  relative  potentials  of  the  two  systems,  since  whether  any  sub- 
stance can  transfer  energy  to  another  depends,  not  on  the  total 
amount  of  energy  in  the  two  substances  or  systems,  but  on  the  poten- 
tial of  the  energy  in  the  two  cases.  The  action  of  any  salt  solution  is 
then  determined  by  its  available  energy,  and  by  the  available  energy 
in  any  salt  is  meant  the  product  of  the  difference  of  potential  between 
the  protoplasm  and  the  salt  multiplied  into  the  amount  of  energy 
transferred  from  one  to  the  other  before  the  potential  is  equalized. 
If  the  protoplasm  and  the  ion  have  energy  at  the  same  potential,  the 
difference  in  potential  will  be  zero,  the  available  energ)'  is  hence  zero, 
the  work  done  is  zero,  and  the  ion  should  produce  no  direct  effect  due 
to  its  energy  content  on  protoplasm,  though  it  might  affect  it  cata- 
lytically  in  the  manner  indicated. 

2.  Total  free  energy. — The  total  free  or  available  energ)'  of  any  ion 
is  composed  of  two  factors,  the  potential  energy  and  the  kinetic  energy. 
The  kinetic  energy,  or  energy  of  motion,  will  be  equal  to  h  MV.  As 
I  do  not  know  the  actual  velocity  of  ionic  movement  when  the  poten- 
tial gradient  is  unknown,  I  am  unable  to  determine  the  kinetic  energy. 
It  is,  in  any  case,  generally  small  when  compared  to  the  potential 
energy,  although  not  negligible  when  the  latter  factor  approaches  zero. 
That  is,  if  the  potential  of  two  ions  and  the  protoplasm  are  about  the 
same,  these  ions  may  have  different  actions  owing  to  differences  in  their 
kinetic  energy,  i.  c.,  their  ionic  masses  and  velocities.  In  this  paper, 
however,  I  shall  consider  only  the  potential  cnerg>'  factor. 


88  A.  P.  Mathews  - 

3.  The  potential  energy  0}  ions. — What  is  the  measure  of  the  poten- 
tial energy  of  any  ion  ?  The  potential  energy  must  be  the  difference 
in  the  energy  content  of  the  ion  or  atom  in  different  conditions.  If 
any  substance  has  any  available  potential  energy,  it  necessarily  means 
that  it  is  capable  of  existing  in  two  conditions  which  differ  in  their 
energy  content,  and  that  it  gives  up  energy  in  passing  from  one  con- 
dition to  the  other.  That  ions  and  atoms  do  exist  in  such  different 
conditions  is  well  known.  Thus  the  chemical  differences  between 
atomic  and  ionic  sodium,  and  between  ferric,  ferrous,  and  metallic 
iron,  are  due  to  differences  in  the  energy  content  of  the  atoms  in  differ- 
ent conditions.  The  available  potential  energy  of  the  sodium  atom 
is  very  much  greater  than  that  of  the  sodium  ion,  as  is  indicated  by 
the  fact  that  when  the  atom  becomes  an  ion,  a  large  amount  of  heat 
is  set  free. 

The  potential  energy  of  the  ion  must  be  sharply  distinguished  from 
the  ionic  potential.  The  potential  energy  is  in  its  turn  composed  of 
a  capacity  and  an  intensity  factor;  the  capacity  factor  being  repre- 
sented by  the  amount  of  electricity  transferred;  the  intensity  fac- 
tor, by  the  tendency  of  the  ion  or  atom  to  change  its  state ;  in  other 
words,  by  its  stability  or  ionic  potential.  The  potential  energy  of 
any  ion  must  be  measured  hence  by  the  ionic  potential  multiplied 
into  the  capacity.  The  capacity  factor  falls  out  of  account  if  equiva- 
lent solutions  are  compared,  since  in  that  case  each  equivalent  has 
the  same  quantity  of  electricity  in  it,  and  the  differences  between  the 
actions  of  ions  are  hence  due  to  differences  in  ionic  potential.  The 
question  now  comes  down  to  the  determination  of  the  ionic  potential. 

4.  The  determination  0}  the  ionic  potential. — In  my  earlier  papers 
it  was  not  clear  to  me  how  this  ionic  potential  could  be  determined, 
so  I  used  instead,  as  a  rough  measure  of  it,  the  solution  tension  of  the 
metal.  I  assumed  that  the  solution  tension  would  be  the  reciprocal 
of  the  ionic  potential.  Inasmuch  as  the  solution  tension  varies  with 
the  concentration  of  the  salt,  I  used  arbitrarily  the  solution  tension 
of  the  metals  in  normal  ionic  solution.  « 

Inasmuch  as  the  determination  of  the  ionic  potential  depends  on 
the  determination  of  the  solution  tension,  it  is  necessary  to  under- 
stand the  latter  term,  and  as  this  may  not  be  familiar  to  all  physiol- 
<5gists,  the  following  explanation  is  given : 


Pharmacodynamics  of  Salts  and  Drugs  89 


TABLE   I. 

Solution  Tension  ; 

[N  Volts  of  Elements  in 

Normal  Ionic  Soldtions. 

Cations 

Anions 

K         -2.92 

Zn     —  0 . 493 

CI       —1.694 

Na       —2.54 

Cd     —0.143 

Br      —1.270 

Ba       -2.54 

Fe"    —0.063 

I         -0.797 

Ca    l-'-'^ 
^^    \-i.88(?) 

Co     +0.045 

NO3— 2.229 

Ni      +0.049 

Sr         -2.49(?) 

Pb     +0.129 

Li        -2.32 

H      +0.277 

^«  {:;::?(.) 

Fe"'  +  o.3i4(?) 

Cu     +0.606 

Al         —0-999 

Hg     +1.027 

Mn      —0.798 

Ag     +1.048 

When  a  plate  of  metal  is  placed  in  water,  a  certain  amount  of  the 
metal  passes  into  the  water  in  the  form  of  positively  charged  par- 
ticles, so  that  the  solution  becomes  positively  charged,  the  metal 
negatively  charged.  The  tendency  of  different  metals  to  throw  off 
these  positive  particles  varies,  and  this  tendency  may  be  measured 
in  so  many  volts  if  the  metal  is  placed  in  a  solution  of  one  of  its  salts 
of  known  strength.  If  this  is  done  and  the  difference  in  voltage 
between  the  metal  and  solution  is  measured,  one  obtains  a  series  of 
values  for  the  different  metals,  and  these  values  are  known  as  the 
solution  tension  series  of  the  metals.  The  measurements  are  gener- 
ally referred  to  the  metals  when  immersed  in  normal  ionic  solutions 
of  their  salts  (Table  i). 

By  a  reference  to  Table  i  showing  this  series  it  will  be  found  that 
potassium  and  sodium  stand  at  one  end  of  the  series,  these  metals 
having  in  normal  ionic  solutions  a  negative  voltage  of  2.92  and  2 .  54 
respectively,  as  compared  with  the  solution;  while  at  the  other  end 
are  the  noble  metals,  gold,  silver,  platinum,  and  mercur}',  which 
immersed  in  normal  ionic  solutions  become  electropositive  to  the 
extent  of  more  than  one  volt. 

The  solution  tension  measures  therefore  the  difference  in  po- 
tential between  the  solution  which  contains  a  known  quantity  of 
the  ions  of  the  metal  and  the  metal  itself;  and  it  expresses  the 
difference  between  the  tendency  of  the  ion  to  deposit  itself  on  the 
metal  plate,  and  the  tendency  of  an  atom  of  the  plate  to  become  an 
ion.  It  will  be  seen  that  the  values  of  the  solution  tension  depend 
entirely  on  the  concentration  of  the  ions  in  the  solution,  and  the  pres- 
ence of  a  plate  or  particle  of  the  metal.     For  our  purposes,  therefore, 


90  A.  P.  Mathews 

it  is  clear  that  the  ordinary  figures  given  for  the  solution  tension  are 
not  strictly  applicable  to  the  physiological  conditions.  We  introduce 
into  the  salt  solution,  not  a  plate  of  metal,  but  a  particle  of  proto- 
plasm, and  we  wish  to  know  what  is  the  tendency  of  any  ion  in  that 
solution  to  give  up  its  charge  in  whole  or  in  part  to  the  protoplasm. 
The  solution  tension  is  not  therefore  a  proper  measure  of  the  par- 
ticular property  of  the  ion  we  seek. 

In  previous  work,  in  order  to  get  comparable  results,  I  had  to 
adopt  arbitrarily  the  solution  tension  of  the  metals  in  the  normal  ionic 
solutions  of  their  salts,  although  there  was  no  reason  at  all  why  this 
value,  rather  than  the  value  in  any  other  concentration,  should  have 
been  taken. 

It  is  clear  that  what  is  most  important  in  the  ion  in  determining 
its  physiological  action,  and  its  chemical  action  as  well,  is  not  the 
difference  of  voltage  between  a  plate  of  metal  and  any  solution  of  its 
salts,  but  rather  the  difference  in  pressure  between  a  single  ion  and  a 
single  atom  of  the  metal.  That  is,  it  is  the  inherent  tendency  of  any 
ion  in  any  concentration  to  change  into  an  atom  of  its  metal.  This 
last  property  has  been  called  the  ionic  potential.  The  method  of  com- 
puting it  is  as  follows : 

Nernst'  has  shown  that  the  formula  which  expresses  the  amount 
of  work  necessary  to  compress  a  gas  from  volume  i  to  volume  2  is  of 
very  general  applicability,  and  also  expresses  the  amount  of  work 
necessary  to  transform  one  gram  atom  of  a  metal  into  one  gram  ion 
at  any  concentration.     This  formula  is  as  follows: 

v 
Amount  of  work  — L  =  RTln—  . 

V2 

In  this  formula  R  is  the  gas  constant;    T,  the  absolute  temperature; 
v^  and-y^  the  gas  volumes ;  and  the  logarithm  is  the  natural  logarithm 
This  formula  may  also  be  expressed  using  pressures  instead  of  vol- 
umes, or: 

L  =  RTln^  . 
Pi 

In  this  formula  p^  is  what  is  known  as  the  solution  pressure  of  the 
metal,  and  p^  the  osmotic  pressure  of  the  ions  of  the  metal  in  the  solu- 
tion.    Instead  of  p^  we  may  write  P.     By  taking  R  and  T  in  absolute 

'  Nernst,  Theoretische  Chemie,  1903,  4te  Aufl. 


Pharmacodynamics  of  Salts  and  Drugs  91 

units,  and  remembering  that  in  all  cases  one  gram  ion  carries  wX  96,540 
coulombs  of  electricity,  where  n  is  the  valence,  this  formula  may  be 
expressed  in  the  form  of  potential  in  volts  existing  between  the  metal 
and  solution,'  i.  e.: 

RT    p,    0.057  /»2 

n      P         n  r 

If  this  potential  is  measured  directly  by  connecting  the  metal 
immersed  in  a  solution  of  its  salt  through  a  voltmeter  with  an  elec- 
trode of  known  potential,  E  may  be  measured,  and  then  P  is  easily 
calculated.  When  P  is  once  known,  E  may  be  calculated  when  the 
metal  is  immersed  in  any  solution  of  its  salts  of  which  p^  is  known. 

To  determine  the  real  ionic  potential  from  this  formula,  one  pro- 
ceeds as  follows :  It  is  obvious  that  the  formula  expresses  the  amount 
of  work  done  in  accomplishing  two  different  things.  It  expresses  the 
sum  of  the  work  necessary  to  transform  one  gram  atom  of  metal  into 
one  gram  ion  in  the  same  space,  plus  the  amount  of  work  (negative) 
necessary  to  expand  the  one  gram  ion  from  this  space  to  one  liter  or 
the  space  it  finally  occupies.  It  is  the  first  of  these  factors  which  we 
wish  to  determine,  since  this  measures  the  ionic  potential.  The  for- 
mula may  accordingly  be  written  as  follows: 

L  =  RTln^  +  RTln^  . 

In  this  formula  p^  is  the  osmotic  pressure  of  the  positive  ions  of  the 

metal  when  at  the  same  concentration  as  the  atoms  of  the  metal;  and 

pj  is  the  osmotic  pressure  of  the  ions  when  at  the  concentration  of  one 

gram  ion  to  the  liter.     Accordingly,  the  first  term  of  the  right-hand 

member  of  the  equation  measures  the  work  necessary  to  transform 

one  gram  atom  of  the  metal  into  one  gram  ion  occupying  the  same 

space,  and  the  second  term  measures  the  work  done  (negative)  in 

expanding  from  this  space  to  one  liter.     Expressing  this  formula  in 

volts,  and  putting  C  =  concentration  in  place  of  p,  and  passing  to 

common  logarithms 

„     0.057  .       ^2  ,  OOS7  1      ^3  /  N 

E  = ^  log -7^  + —  log-^  .  (i) 

n  C         n  L\ 

In  this  equation  E  is  determined  by  measurement,  and  the  second  term 
is  easily  calculated.     The  middle  term,  or  the  ionic  potential,  is  then 

»  Ibid.,  p.   701. 


92  A.  P.  Mathews 

obtained  by  the  difference  between  E  and  the  second  term.  For 
example,  a  silver  plate  in  contact  with  a  normal  ionic  silver  nitrate 
solution  shows  a  difference  of  potential  between  itself  and  the  solution 
of  +1.048  volts.  .'.£=+1.048  volts.  C2  is  the  concentration  of 
silver  atoms  in  metallic  silver.    One  gram  atom  of  silver — i.  e.,  107 .9 

TABLE  2. 

The  Ionic  Potentials  of  the  Ions  of  Metals  in  Volts. 

K    -2.92(?)  Cd    -0.089  O -0.426 

Na-2.54(?)  Fe"  +  o.oo  Cl-i. 694(7) 

Li   — 2.32(?)  Co   +0.107  '        Br  — i.27o(?) 

Ba  -2.54(?)  Ni    +0.112  I    -o.797(?) 

Ca  -2.26(?)  Pb   +0.179 

Sr   -2.      (?)  H     +o.io7(?) 

Mg  - 1 . 1 60  Cu    +  o .  668 

Mn— 0.737  Hg  +1.080 

Zn  -0.434  Ag    +1.163 

grams  of  silver — occupies  the  space  at  i8°  of  lo.  i  c.c. ;  i.e.,  the 
atomic  weight    in   grams    divided  by   the    specific  gravity,     c^,  or 


1000 


the  number  of  gram  atoms  of  silver  in  one  liter  of  silver,  = .      c 


10. 1 


3 


=  1,  since  the  concentration  of  silver  ions  is  one  gram  ion  per  liter. 
Substituting  these  values  in  (i), 

10. 1 


1 .048  volts  =  o.o57  log  7^  +  0057  log 

C  1000 


or 


1.163  =  0.057  log^  . 

There  is  hence  a  difference  of  potential  of  i .  163  volts  between  one 
atom  of  silver  and  one  ion,  in  favor  of  the  ion.  That  is,  when  one 
gram  ion  of  silver  changes  into  one  gram  atom  in  the  same  space 
96,540  X 1 .  163  Joules  of  energy  are  set  free,  or  since  each  monovalent 
ion  carries  9.65X10""^°  coulombs  of  electricity,  when  one  silver  ion 
changes  into  a  silver  atom  at  any  concentration,  9.65X  io~^°X  i .  163 
Joules  of  energy  are  set  free. 

It  will  be  seen,  by  comparing  Tables  i  and  2,  that  the  true  values 
of  the  ionic  potential  calculated  in  this  way  do  not  in  most  instances 
differ  greatly  from  the  values  of  the  solution  tension  in  normal  ionic 
solutions.  The  heavy  metals  have,  as  a  rule,  an  ionic  potential  about 
0.07—0.1  volts  different  from  the  solution  tension  in  normal  ionic 
solutions.* 

*  I  have  not  calculated  the  ionic  potentials  of  CI.  Br,  and  I,  but  have  used  instead  the  figures  for  solu- 
tion tension  in  normal  ionic  solution.  It  is  also  impossible  to  calculate  the  ionic  potentials  of  Ca,  Li,  Ba, 
Na,  and  K. 


Pharmacodynamics  of  Salts  and  Drugs  93 

A  word  may  be  said  about  the  reliability  of  these  figures.  They 
depend,  as  will  be  seen,  upon  direct  measurements  of  the  electromo- 
tive force  shown  between  two  metals  when  immersed  in  known  solu- 
tions of  their  salts,  those  solutions  being  in  contact.  This  assumes  a 
knowledge  of  the  number  of  ions  of  metal  in  the  salt  solutions  in  ques- 
tion, and  this  factor  is  not  in  all  cases  perfectly  certain.  A  much  more 
serious  source  of  possible  error  arises  from  a  determination  of  the 
potential  of  any  single  metal,  since  it  is  necessary  to  know  the  poten- 
tial of  at  least  one  electrode  before  the  rest  can  be  determined.  The 
measurement  is  ordinarily  made  by  using  the  calomel  mercury  elec- 
trode, which  is  supposed  to  have  a  potential  of  +0.56  volts.  This 
voltage  was  determined  by  measuring  the  potential  between  a  drop- 
ping mercury  electrode,  which  theoretically  should  have  a  zero  poten- 
tial, and  the  calomel  mercury  electrode.  Recent  determinations  of 
the  potential  of  the  calomel  mercury  electrode  by  a  totally  diflferent 
method  by  Billitzer'  give  a  different  value.  It  is,  therefore,  impos- 
sible at  present  to  say  which  of  these  measurements  or  methods  gives 
the  more  reliable  result.  Nernst  has  accordingly  proposed  that  the 
hydrogen  electrode  in  normal  ionic  solution  be  regarded  arbitrarily 
as  zero,  until  the  absolute  potential  is  determined.  I  have,  however, 
used  the  values  as  given  by  Wilsmore,  based  on  the  calomel  electrode 
0.56. 

The  question  does  not  influence  most  of  the  measurements  which 
follow,  since  these  are  based  on  the  sum  of  the  potentials  of  both 
anion  and  cation,  or  upon  the  differences  between  two  like  ions. 
This  is  a  constant  whatever  the  absolute  potential,  since  if  a  certain 
number  is  added  to  the  anion,  it  will  be  subtracted  from  the  cation. 
For  example,  suppose  it  be  shown  by  a  change  in  the  point  of  zero 
potential  that  the  solution  tension  of  sodium  is  2 .  34  instead  of  2 .  54, 
this  increases  the  ionic  potential  by  0.2  volt;  but  if  sodium  is  2.34, 
then  chlorine  is  i .  894  instead  of  i .  694,  and  this  reduces  the  ionic 
potential  of  chlorine  by  0.2  volt.  The  sum  of  the  potentials  of 
sodium  and  chlorine  remain  unchanged. 

b)  The  relation  between  physiological  action  and  ionic  potential. — 
To  bring  out  the  relationship  between  ionic  potential  and  toxic  action, 
I  have  prepared  Table  3,  which  shows  this  relationship  for  the  toxic 

■  BiLtrrzER,  Ztschr.  fur  Eiektrochemie,  1902,  8,  p.  638. 


94 


A.  P.  Mathews 


action  of  salts  on  fish  eggs/  the  diastatic  ferment,^  bromelin,^  a  pro- 
teolytic ferment,  and  growing  tips  of  peas  and  beans/  Table  3  shows 
that  the  chlorides  of  the  various  metals  arrange  themselves,  as  regards 
their  toxicity,  with  few  exceptions,  in  the  order  of  the  potential  energy 
of  their  ions.  Ions  of  low  potential  energy,  such  as  those  of  sodium, 
lithium,  magnesium,  and  potassium,  being  relatively  inert,  when  com- 
pared with  the  enormously  toxic  action  of  the  ions  of  high  potential 
energy,  such  as  nickel,  lead,  hydrogen,  ferric,  cupric,  mercury,  silver, 
gold,  and  platinum. 

There  is,  I  think,  no  mistaking  this  general  parallelism,  which  was 
theoretically  anticipated.  By  no  other  properties  known  to  us  can 
the  metals  be  arranged  in  an  order  so  closely  corresponding  to  their 

TABLE  3. 

Minimum  Fatal  Doses  of  Salts  for  Various  Ferments  and  Organisms. 

(F  =  Dilution  Minimum  Fatal  Dose  (Equivalent).) 


SALT 


Ag  NO3 

HgN04 

Hg  Ch 

Hg(CN),   

Cu  SO4 

Cu  Cla 

TeCla 

Pb  (N03)> 

Pb  Ch 

Pb  (CHjO,),.. 

HCl 

Cd  (NO3), 

CdCU 

Ni  CI, 

NiSO^ 

CoCh 

Co  SO4 

Co  (NOj), 

Fed, 

FeSO* 

Zn  Cl 

Zn  (NO,), 

Zn  SO4 

Mn  Ch 

AICI3 

MgCU 

Mg  (NO3), 

Li  Cl 


CaCl... 
Ba  CU.. 
Li  Cl,... 
NaCI... 
KCl.... 
Na  NO3. 
KNO3.. 


Minimum  Fatal  Dose  (V)  Equivalent  Dilution 


Diastase 


<  100,000 
30,000 


8,333 
333 
3o(?) 


990 


142 
910 


69 


6.25 

3-3 

I 


1-4 

4 

1. 1 

2-5 

>  3 

>  3 


Eggs  of 

Cilia 

Bromelin 

Roots  of 

Roots  of 

Fundulus 

Volvox 

Pisum 

Zea  mais 

100,000 

300,  x>o 

110,000 
75,000 

204,800 

300,000 

50,000 

204,800 

25,600 

30,000 

51,200  ■ 

102,400 

15,000 



24,000 

51,200 

102,400 

4,000 

20,000 

5,000 

18,500 

5,000 

3,000 

3,000 

12,&O0 

3,200 

12,500 

500 



500 





51,200 

51,200 

250 

2,250 

2,400 

25,600 

6,400 

10 

■  '  800 

14,000 
8,3  so 

4 

IS 

3 

2 

S 

32s 

4 

1,000 
(Licl) 

3-5 

5 

2 

i-S 

400 

2 

1-3 

4(?) 

100 

i,6oo(  ?) 

100 

Roots  of 
Lupinus 


300,000 


51,200 
12,800 
12,800 

6,700 


3,200 
51,200 


12,800 

12,800 
12,800 

6,400 


'Authors'  results. 
'  McGiugan,  luc.  cit. 


3  CaldweU. 

*  Kahlenberg  and  True,  loc.  cit.;  rieald,  loc.  cu 


Pharmacodynamics  of  Salts  and  Drugs 


95 


toxic  action.  For  example,  suppose  it  was  attempted  to  classify  them  in 
the  order  of  their  ionic  weights.  It  will  be  seen  that  no  parallelism 
exists  between  toxic  action  and  ionic  weight,  since  we  have  nickel,  atomic 
weight  58,  lead,  atomic  weight  200,  hydrogen,  atomic  weight  i,  and 
copper  atomic  weight  32,  following  each  other  closely.  Furthermore, 
attention  may  be  called  to  the  enormous  difference  in  toxicity,  exist- 
ing between  the  same  atom  when  carrying  two  different  quantities  of 
potential  energy.  Ferrous  iron  has  as  an  ion  very  little  potential  energy 
compared  with  ferric  iron,  and  it  is  enormously  less  poisonous  than  the 
latter. 

It  will  be  noticed  also  that  in  each  of  these  cases  certain  metals 
come  out  of  their  proper  order  of  toxicity  and  potential.  In  the  case 
of  Fundulus  eggs,  cadmium  is  considerably  out  of  its  proper  place, 
whereas  it  follows  the  rule  in  the  case  of  diastase;  for  diastase,  lead  is 
quite  out  of  its  position;  for  bromelin,  the  great  exception  is  barium, 
which  is  very  toxic.  The  causes  of  these  special  and  sporadic  excep- 
tions will  be  taken  up  later. 

TABLE  4. 

MiNiuuM  Fatal  Dose  and  Ionic  Potential  of  Anions. 

(Eggs  of  Fundulus  heleroclitus.) 


Salt 

V  (Min.  Fatal  Dose) 

Anionic    Potential 

NaNo. 

NaCl 

2.0 
2.0 

2-7 

4.0 
11 .0 
3S-0 

—  2.229  ( ?) 
-1.694    " 

—  1.270    " 
-0.797    " 
-0.727    " 
-0.109   " 

NaBr 

Nal 

NaBrOj 

Na.C,04 

If  now  we  turn  to  the  negative  ions,  or  anions,  a  similar  paralklism 
is  shown  to  exist  between  toxicity  and  potential  energy  content.  Thus 
chlorine  is  in  all  cases  far  less  toxic  than  iodine,  which  has  twice  as 
much  potential  energy.  The  oxalates  and  cyanides  and  sulphites  are 
the  more  toxic,  the  greater  their  available  energy  content.  Unfortu- 
nately, our  knowledge  of  the  potentials  of  the  anions  is  less  exact  than 
our  knowledge  of  the  potentials  of  the  cations,  so  that  it  is  impossible 
to  follow  the  correspondence  in  detail;  but  sufficient  is  shown  to  prove 
that  the  same  correspondence  between  toxicity  and  potential  energy 
exists  here  as  in  the  cations. 

c)  The  quantitative  relationship  between  ionic  potential  and  the 
minimum  fatal  dose. — It  was  shown  at  the  outset  that  the  amount  of 


96 


A.  P,  Mathews 


work  any  ion  can  do  must  depend  upon  its  available  potential  energy ; 
i.  e.,  upon  the  product  of  the  difference  of  potential  between  the 
protoplasm  and  the  ion  multiplied  into  the  quantity  of  energy 
transferred  before  the  potentials  were  equalized.  Of  two  positive 
ions  holding  different  quantities  of  available  potential  energy,  that 
which  has  the  more  energy  can  do  the  more  work.  The  total  amount 
of  work  any  number  of  positive  ions  can  do  will  depend  on  the  con- 
centration of  the  the  ions  and  amount  of  available  potential  energy  in 
each  ion.     If  we  take  as  a  standard  a  certain  amount  of  work — let  us 


o 
Q 


Q 


X  =  Ionic  Potential. 

Fig.  I.     Ordinates  represent  the  dilution  of  the  minimum  fatal  dose 
Abscissae  represent  the  cation  potential. 


Pharmacodynamics  of  Salts  and  Drugs  97 

say  the  amount  of  work  just  sufficient  to  kill  protoplasm  in  unit 
time,  say  24  hours — the  concentrations  of  the  two  ions  which  will 
just  accomplish  this  work  must  stand  in  some  numerical  relation  to 
their  potential  energy  content.     What  is  that  relation  ? 

To  bring  out  this  relationship,  I  have  plotted  the  curve  in  Fig.  i, 
which  expresses  the  relationship  between  the  ionic  potential  of  the  cation 
and  the  dilution  (V)  of  the  minimum  fatal  dose.  By  dilution  is  meant 
the  number  of  Hters  of  solution  containing  one  gram  equivalent  of  the 
salt.  From  an  inspection  of  this  curve  it  will  be  seen  that  we  are 
dealing  with  a  logarithmic  function;  that  is,  the  dilution  increases 
enormously  in  a  logarithmic  ratio  to  the  ionic  potential.  The  dilution, 
for  example,  increases  100,000  times  while  the  ionic  potential  increases 
about  five  times. 

By  inspection  of  this  curve  one  may  write  the  general  equation : 

log,^V=KE  .  I 

In  this  formula  E  is  the  ionic  potential,  V  the  dilution,  and  K  the  con- 
log  V 
stant  of   proportion.     If,  however,  we   place  — pr-  =  K,    it  will  be 

seen  that  this  formula  is  not  in  the  right  form,  since  E  for  some  ions  is 

1?- 

zero.     Nor  does  the  formula  log  V=  ^   1  f   give  constant  results.    In 

this  formula  E'^  +  E"  represents  the  sum  of  the  potentials  of  the  anion 
and  cation.  Nor  could  it  be  anticipated  that  such  a  formula  would 
give  a  proper  result,  since  what  we  have  to  express  is  the  difference  of 
potential  between  the  salt  and  the  protoplasm,  and  this  formula 
expresses  only  the  relation  between  minimum  fatal  dose  and  the 
absolute  potential  of  the  various  ions.  Before  setting  up  any  theo- 
retical formula,  it  is  necessary  to  get  clearly  in  mind  just  in  what  the 
difference  in  potential  between  the  protoplasm  and  the  salt  consists. 
d)  Derivation  oj  a  theoretical  formula  expressing  the  relationship 
between  minimum  fatal  dose  and  the  available  potential  energy  of  salt 
solutions. — We  have  now  found  out  how  to  measure  the  potential 
of  the  potential  energy  of  the  salt  solution;  it  is  necessary  that  we 
discover  also  how  the  potential  of  the  potential  energy  of  the  proto- 
plasmic system  is  to  be  determined,  since  our  formula  involves  both 
these  factors,  the  available  potential  energy  being  the  difference  be- 
tween the  potentials  of  the  salt  and  the  protoplasm. 


98  A.  P.  Mathews 

The  protoplasmic  system  is  made  up  cf  masses  of  proteid  matter 
in  equilibrium  apparently  with  particles  of  the  same  proteid  in  solution. 
It  may  be  regarded  as  a  two-phase  colloidal  system.  We  may  assume, 
in  the  light  of  the  investigations  of  the  past  five  years,  that  changes  in 
the  protoplasmic  activity  are  due,  in  part  at  least,  to  changes  in  the 
state  of  these  colloidal  particles  and  masses,  and  that  the  salts  are 
affecting  vital  processes  in  part  by  producing  such  changes.  What 
we  have  to  compare,  then,  in  the  first  instance,  is  the  potential  of  the 
energy  of  the  protoplasmic  colloids  with  the  potential  of  the  potential 
energy  of  the  ions  of  the  salt  solution.  We  have,  therefore,  to  get  a 
clear  idea  of  the  relationship  of  salts  to  the  precipitation  and  solution 
of  colloids. 

Since  the  protoplasmic  colloids  are  for  the  most  part  composed  of 
albumin  in  combination  with  other  radicles,  it  is  to  the  albuminous  or 
proteid  colloids  to  which  attention  may  first  be  directed.  The  work 
of  Kossel  and  Fischer  has  cleared  up  the  structure  of  the  albumin 
molecule,  which  has  been  shown  to  be  a  polymer  of  amino  acids.  As 
a  result,  albumin  or  proteid  is  shown  to  be  both  acid  and  basic;  that  is, 
it  is  capable  of  uniting  with  metals  to  form  true  salts,  and  also  with 
acids  through  the  amino  group. 

If  common  egg  albumin  is  dissolved  in  alkaline  solution,  it  exists 
as  sodium  albuminate;  if  it  is  dissolved  in  hydrochloric  acid,  it 
exists  as  albumin  chloride.  Sodium  albuminate  dissociates  electro- 
lytically,  owing  to  the  high  dissociating  power  of  sodium  (that  is,  its 

high  solution  tension),  and  Na  +  and  albumin  ions  are  formed.  Simi- 
larly, owing  to  the  high  dissociating  power  of  the  chlorine,  albumin 

+  

chloride  dissociates  into  albumin  and  CI  ions.      This  dissociation 

results  in  giving  the  albumin — that  is,  the  colloidal  particle — a  positive 
or  negative  electric  charge.  It  is  possible  to  change  the  sign  of  the 
charge  on  the  albumin  particles  by  making  an  alkaline  solution  suffi- 
ciently acid.  This  is  brought  about  in  the  following  way :  By  adding 
acid  to  the  alkaline  albumin  the  highly  dissociated  sodium  compound 
is  replaced  by  the  slightly  dissociated  hydrogen  compound.  The 
result  of  this  is  that  the  charge  on  the  colloid  is  neutralized,  undis- 
sociated  albumin  is  formed,  and  if  the  concentration  of  the  albumin  is 
sufficiently  great,  precipitation  will  occur.     If  one  continues  to  add 


Pharmacodynamics  of  Salts  and  Drugs  99 

acid,  the  albumin  combines  with  the  hydrochloric  acid  and  goes  over 
into  the  chloride.  This  at  once  dissociates  the  chlorine  ion  as  a  nega- 
tive ion,  and  the  albumin  becomes  the  cation.  It  will,  however,  be 
clear  that,  although  in  this  case  the  albumin  is  mainly  electropositive, 
yet  it  is  an  acid,  and  the  ionization  of  its  hydrogen  is  not  completely 
suppressed,  although  it  is  reduced  to  a  very  small  amount.  This 
means  that  here  and  there  are  albumin  particles  which  are  at  one  spot 
electronegative,  since  they  dissociate  hydrogen,  and  in  another  spot 
electropositive,  since  they  dissociate  chlorine.  We  have  some  am- 
photer  or  twin  ion  colloids,  in  other  words.  The  evidence  of  the 
existence  of  such  ions  will  be  taken  up  on  p.  104. 

I  make  this  explanation  at  such  length  because  it  does  not  appear 
to  be  clear  to  many  that  the  albumin  colloids  owe  their  charges  to 
processes  of  ionization,  just  as  any  salts  owe  their  charges  to  these 
processes.  Thus  several  writers  have  assumed  that  the  charge  was 
owing  to  the  salt  introduced.  The  ion  of  the  salt  introduced  which 
moved  fastest  was  supposed  to  bury  itself  in  the  colloid  particle,  and 
thus  give  its  charge  to  it.  This  hypothesis  is  quite  unfounded  and 
undoubtedly  erroneous. 

The  proteids,  therefore,  in  protoplasm  exist  as  salts,  and  dissociate 
sodium  or  other  metalHc  ions,  and  chlorine  and  other  anions,  and  the 
colloids  thus  become  charged.  Some  proteids  here  and  there  dis- 
sociate both  positive  and  negative  ions.  The  colloids  in  protoplasm 
are  undoubtedly  in  the  condition  of  a  saturated  solution,  and  we  have 
an  equilibrium  between  dissociated  and  undissociated  colloids,  in 
solution,  and  undissociated  and  dissociated  precipitated  colloids.* 
It  has  been  shown,  furthermore,  that  the  state  of  solution  and  the 
fineness  of  subdivision  of  the  colloidal  particles  depend  on  the  number 
of  free  electrical  charges  on  their  surfaces.  The  greater  the  number 
of  similar  charges,  the  greater  the  solubility  of  the  colloid. 

In  determining  the  potential  of  the  potential  energy  of  the  colloid, 
we  have,  then,  just  the  same  factors  to  consider  as  in  the  salts,  since  the 
colloids  are  salts.  The  potential  of  the  energy  content  of  the  colloid 
solution  must  be  determined  by  the  ionic  potentials  of  the  ions  into 
which  it  dissociates,  for  example,  the  potentials  of  sodium  and  albumin. 

*As  a  possible  example  of  a  dissociated  insoluble  colloid,  fibrin  which  has  been  in  acid  may  be  men- 
tioned. This  fibrin  dissociates  hydrogen,  but  the  hydrogen  ion  is  unable  to  move  away  from  the  fibrin. 
The  condition  is  very  similar  to  the  double  layer  at  the  surface  of  an  electrode. 


loo  A.  P.  Mathews 

In  studying  the  action  of  any  salt  on  an  albumin  solution,  the  real 
question  to  be  answered  is:  What  will  be  the  result  upon  the  solu- 
bility and  state  of  the  colloid  of  replacing  the  ion  already  in  combina- 
tion with  the  proteid  by  some  other  ion  containing  a  different  amount 
of  potential  energy  ?  We  have  to  know,  therefore,  before  we  can 
answer  the  question  of  the  action  of  any  salt  on  a  colloid,  what  the  ion 
is  which  is  already  in  combination  with  it.  This  is  a  very  important 
point,  which  is  frequently  overlooked  in  studying  the  action  of  salts. 

To  get  a  clear  idea  of  what  happens  when  a  salt  is  added  to  an 
albumin  solution,  let  us  consider  first,  the  condition  of  affairs  in 
the  sodium  albumin  solution  in  which  the  proteid  exists  as  Na  +  albumi- 
nate. As  the  colloid  stands  in  a  saturated  solution,  it  is  in  a  condition 
of  equihbrium.  The  sodium  ion  has  separated  a  certain  distance 
from  the  albumin  ion.  The  distance  it  moves  depends,  no  doubt,  on 
several  factors,  but  the  most  important  will  probably  be  its  tendency 
to  go  into  solution — i.  e.,  its  solution  tension.*  The  positive  ion  of 
sodium  repels  a  negative  charge  with  a  power  equal  to  2.54  volts. 
What  the  negative  solution  tension  of  the  albumin  ion  is,  unfortunately 
is  unknown.  The  effect  of  the  positive  charge  on  the  sodium  will 
be,  of  course,  to  neutrahze  the  negative  charge  on  the  albumin, 
but,  owing  to  the  fact  that  the  sodium  repels  the  negative  charge  and 
holds  its  positive  charge  so  firmly,  it  is  unable  to  neutralize  it,  and  the 
colloid  remains  in  solution.  We  have,  in  other  words,  an  equilibrium 
between  dissociated  and  undissociated  sodium  albuminate. 

The  question,  then,  to  be  solved  is  this :  What  effect  will  it  have  on 
the  solubility  of  the  colloid  if  we  replace  the  sodium  ion  by  another 
ion  containing  a  different  quantity  of  potential  energy,  i.  e.,  having  a 
different  ionic  potential  ?  One  of  two  results  may  be  anticipated : 
either  the  dissociation  will  increase  and  the  colloid  go  more  completely 
into  solution,  or  it  will  diminish  and  the  colloid  be  more  or  less  com- 
pletely precipitated. 

If  we  introduce  an  ion  of  higher  potential  than  sodium,  evidently 
the  state  of  equihbrium  can  no  longer  be  the  same.  Energy  will 
pass  from  the  positive  ion  to  the  albumin,  and  will  in  some  degree  hold 
or  neutralize  its  negative  charge.      We  may  imagine  that  the  new 

*Many  facts  indicate  that  one  of  the  most  important  factors  in  determining  the  ionization  of  salts 
is  the  ionic  potential  of  its  ions.  For  example,  compare  the  ionization  constants  of  the  iodide,  chloride, 
and  bromide  of  mercury  or  silver;  or  compare  the  ionization  of  silver  and  sodium  nitrates. 


Pharmacodynamics  of  Salts  and  Drugs  ioi 

ion  no  longer  can  move  so  far  from  the  albumin,  and  in  consequence 
more  nearly  neutralizes  its  charge.  The  result  is  that  the  surface  of 
the  colloidal  particles  will  be  reduced,  the  surface  tension  will  be 
increased  and  the  colloid  will  be  less  stable.  In  another  way  of 
putting  it,  the  dissociation  is  somewhat  reduced  and  consequently 
some  of  the  colloid  tends  to  precipitate.  If,  in  fact,  the  ion  used  to 
supplant  the  sodium  has  a  sufficiently  high  potential,  it  will  practically 
not  leave  the  colloid  at  all,  the  dissociation  will  be  greatly  reduced,  the 
negative  charges  almost  neutralized,  and  precipitation  will  occur. 
If  the  ionic  potential  of  the  introduced  ion  is  still  higher,  it  may  oxidize 
the  colloid;  i.  e.,  an  actual  exchange  of  charges  will  take  place  between 
the  albumin  and  the  ion. 

If,  however,  an  ion  of  lower  potential  is  introduced  in  place  of  the 
sodium,  the  reverse  of  these  processes  will  take  place.  Ionization 
will  be  increased,  the  negative  charge  will  be  freer,  and  the  solubility 
of  the  colloid  will  be  greater.  This  will  be  the  case  if  potassium  is 
substituted  for  the  sodium,  provided  that  potassium  has  a  lower  ionic 
potential  than  sodium,  as  is  generally  assumed,  and  that  no  other 
factors  come  into  play. 

It  is,  therefore,  clear  that  the  effect  of  any  salt  upon  a  saturated 
colloidal  solution  of  albumin  in  which  the  albumin  is  electronegative 
will  depend  chiefly  upon  what  ion  is  in  combination  with  the  colloid 
when  the  salt  is  introduced. 

The  quantitative  differences  in  the  effects  of  different  salts  must 
depend  upon  the  differences  in  the  ionic  potentials  of  the  ion  in  com- 
bination with  the  proteid  and  that  substituted  for  it. 

So  far,  we  have  considered  only  the  role  of  the  positive  ion.  That 
of  the  negative  ion  is  also  of  importance,  but  somewhat  more  diffi- 
cult to  picture  to  ourselves.  We  may,  however,  look  at  it  in  this 
way.  The  different  negative  ions  introduced  have  different  ten- 
dencies to  deposit  on  the  colloid,  and  give  up  their  negative  charges 
to  it.  This  tendency  is  measured  by  the  ionic  potential  of  the  ion. 
If  the  negative  ion  does  deposit,  it  will  tend  to  increase  the  negative 
charge  on  the  colloid,  and  hence  to  dissolve  it.  The  higher  the  ionic 
potential  of  the  anion  introduced,  the  greater  must  be  its  dissolving 
action  on  the  colloid,  since  the  greater  will  be  its  tendency  to  give  its 
negative  charge  to  the  albumin.     If  the  ionic  potential  of  this  ion  is 


I02  A.  p.  Mathews 

lower  than  the  albumin,  it  will  have  an  opposite  or  precipitating 
action,  since  then  the  colloid  will  tend  to  give  up  its  charge  to  it. 

These  conclusions  are  confirmed  by  my  results  on  sodium  albumi- 
nate, and  those  of  Osborne  and  Harris  on  edestin. 

From  these  general  considerations  the  conclusion  may  he  drawn  that 
the  precipitating  action  of  the  salt  on  the  colloid  will  be  proportional  to 
the  difference  between  the  ionic  potential  of  the  positive  ion  already  com- 
bined with  the  colloid  and  that  substituted  for  it;  and  that  the  dissolving 
action  of  the  anion  will  be  proportional  to  the  difference  in  potential  of 
the  anion  of  the  colloid  and  that  we  introduce;   or 

Precipitating  action =£<;  salt— -Ec  coUoid- 
Dissolving  action=£asait— -Ea  coUoid- 
In  this  formula  Ec  salt  is  the  ionic  potential  of  the  cation  of  the  salt 
introduced,  and  E^  coUoid  that  of  the  cation  of  the  colloid.     E^  salt 
and  Ea  coUoid  ^■re  the  values  for  the  anions. 

Since  these  two  actions  are  mutually  antagonistic,  the  actual  action 
of  the  salt  will  be  equal  to  the  difference  between  them,  or 
Actual  action  =  precipitating— dissolving  action 

—  ^c  salt      ^c  colloid      ^a  salt  "•"  ^a  colloid 
=  (£i_£i")  _(£«_£-) 

If  the  result  is  positive,  the  salt  should  precipitate ;  if  it  is  negative, 
it  should  dissolve  the  colloid.  If  it  is  zero,  the  salt  should  not  affect 
the  colloid  except  by  mass  action  or  by  action  on  the  water.  In  other 
words,  the  actual  action  of  any  salt  on  a  colloid  in  solution  will  be  pro- 
portional to  the  difference  between  the  ionic  potentials  of  the  ions  of  the 
salt,  minus  the  difference  in  ionic  potentials  of  the  ions  of  the  colloid. 

Let  it  be  assumed  that  the  logarithm  of  the  dilution  of  the  least 
precipitating  concentration,  or  the  logarithms  of  the  dilution  of  solu- 
tions of  equivalent  dissolving  power,  are  proportional  to  the  actual 
action  of  the  salt.^     This  gives  the  following  equation: 

log  V  =  K[(E'-E^)-(E^-E'')]  +  const.  (2) 

Comparing  two  salts  with  the  same  sign  of  action,  i.  e.,  dissolving  or 
precipitating. 

log  F,  =  Al(£i-£f)-(£"-£'^)]+const. 
log  F,  =  ir[(£i-£f)  -  (£"-£'^)]  +const. 

log  f; = ^[  (M  -  Ef)  -  (4  -  £r)  ]  .  (3) 

■  See  Fig.  I,  p.  q6. 


Pharmacodynamics  of  Salts  and  Drugs  103 

That  is,  the  logarithm  0}  the  ratio  between  equivalent  precipitating  con- 
centrations of  two  salts,  divided  by  the  difference  between  the  differ- 
ences of  potentials  of  the  ions  of  the  two  salts  ought  to  give  a  cofislant* 

Wc  thus  have  for  the  first  time  a  formula  for  application  to  pro- 
toplasm which  states  clearly  at  the  outset  that  the  effect  of  any  salt 
solution  on  the  protoplasm  will  depend  upon  what  ions  are  already 
in  combination  with  the  protoplasm.  In  other  words,  if  we  supplant 
most  of  the  ions  in  any  cell  by  sodium,  and  then  apply  calcium  chloride, 
the  effect  will  be  different  from  that  obtained  if  calcium  chloride  is 
applied  before  the  sodium  chloride.  Furthermore,  the  same  salt  will 
act  differently  on  different  cells,  if  only  those  cells  have  different  ions 
in  them.  Both  of  these  necessary  conclusions  of  the  theory  have 
been  established  by  observation.  To  make  this  perfectly  clear,  the 
difference  in  potential  between  the  protoplasm  and  the  salt  solution 
which  we  started  to  measure  is  the  difference  in  potential  between  the 
systems  ionized  colloid — ionized  salt. 

However,  this  formula  cannot  be  applied  directly  as  it  stands  to 
protoplasm  as  a  whole,  because  it  only  applies  to  colloidal  solutions 
in  which  the  colloids  are  all  of  one  sign.  In  protoplasm,  however,  it 
is  certain  that  we  have  colloids  of  both  signs  and  very  probably 
amphoter  colloids;  i.e.,  colloids  which  are  both  positive  and  nega- 
tive at  different  parts  of  the  molecule. t     We  probably  have,  in  other 

*  This  formula  may,  I  think,  be  substituted  with  advantage  for  that  of  the  tension  coefficient.  It 
is  in  reality  the  numerator  of  the  tension  coefficient. 

t.\  number  of  facts  speak  for  the  presence  of  such  twin  ions  in  colloidal  albumin  solutions  and  in 
protoplasm.  For  example,  if  egg  albumin  is  dialy7,ed  nearly  free  from  salts,  and  then  coagulated  so  as 
to  form  a  weakly  alkaline  colloidal  solution  of  albumin,  and  if  this  solution  is  then  made  acid,  it  is  well 
known  that  the  albumin  becomes  predominantly  electropositive.  That  is  shown  by  the  colloid  migrating 
slowly  to  the  cathode  in  an  electric  field,  and  also  by  the  combining  power  of  the  albumin,  since  it  now 
combines  readily  with  picric  and  other  acids  to  form  albumin  picrate,  tannate,  and  so  on.  Nevertheless, 
if  the  solution  is  not  too  acid,  it  will  combine  still  with  the  metals  in  some  measure.  This  is  undoubtedly 
due  to  the  composition  and  character  of  the  albumin.  The  alkali  albumin  first  obtained  by  heating 
is  a  salt.  When  the  albumin  has  add  added  to  it,  there  is  formed,  in  the  first  instance,  the  free  acid  of 
the  albumin,  which  is  not  much  dissociated.  In  addition,  the  acid  is  added  to  the  amido-group,  and  in 
an  excess  of  acid  hydrolytic  decomposition  being  greatly  reduced,  the  dissociation  takes  place  so  as  to  make 
the  albumin  predominantly  electropositive.  However,  the  ionization  of  the  acid  is  not  entirely  prevented, 
although  it  is  greatly  reduced,  so  that  in  some  places  we  must  have  some  hydrogen  ions  being  formed, 
leaving  the  albumin  electronegative  at  certain  places.  It  is  probably  this  small  percentage  of  hydrogen 
ions  which  can  still  be  replaced  by  the  metals.  I  found  that,  as  a  matter  of  fact,  the  heavy  metals  mer- 
cury and  copper,  although  they  would  not  in  themselves  cause  a  precipitate  if  the  solution  were  sufficiently 
acid,  yet  they  rendered  the  albumin  far  more  easily  precipitated  than  it  was  before.  This  is  to  be  antici- 
pated on  our  \-iew  that  the  colloidal  particles  are  in  some  places  negative  and  in  other  places  positive.  Not 
only  does  this  appear  to  be  the  case  for  albumin,  but  in  protoplasm  there  is  also  reason  for  believing  that 
both  ions  of  the  salt  are  actually  bound  by  the  protoplasm,  and  especially  in  Fundulus  eggs.  It  will  be  re- 
membered that  Du  Bois  Raymond  long  ago  assumed  that  such  polarized  particles  might  exist  in  proto- 
plasm. 


I04 


A.  P.  Mathews 


K 


Cl 


Fig.  2.  Illustrating  a  colloidal  twin  ion 
dissociating  at  one  place  K,  and  at  another  Cl, 
leaving  the  colloid  both  negative  and  positive 
at  different  places. 


words,  ions  like  that  in  Fig.  2.  As  a  matter  of  fact,  if  we  try  to 
apply  this  formula  to  the  results  on  toxicity,  a  discrepancy  between 
the  response  of  protoplasm  and  colloidal  albumin  to  salts  is  at  once 
noticed,  I  have  already  called  attention  to  this  discrepancy.  The 
discrepancy  is  this:  While  in  the  colloidal  solutions  the  opposite 
action  of  the  ions,  dissolving  and  precipitating,  is  clearly  apparent, 

and  that  opposite  action  is  propor- 
tional to  the  ionic  potentials  of  the 
anion  and  cation  respectively,  for 
protoplasm  in  general  and  for  the 
ferment  studied  by  McGuigan  a  dif- 
ferent relationship  is  seen  in  that, 
instead  of  the  positive  ion  counter- 
acting by  its  energy  content  the 
negative  ion,  as  it  ought  to  do  on 
the  theory  developed,  a  summation 
of  effects  is  noticed.  The  iodides  of  the  metals,  instead  of  being 
less  poisonous  than  the  chlorides,  as  they  should  be,  are  more 
poisonous.  The  explanation  of  these  facts  is  to  be  sought  on  the 
basis  of  the  differently  charged  colloids  present. 

If  these  amphoter  colloidal  particles  exist  in  protoplasm,  or  if  we 
are  dealing  in  protoplasm  with  a  mixture  of  both  negative  and  positive 
colloids,  each  ion  will  tend  to  precipitate.  In  the  twin  ions,  if  potas- 
sium is  replaced  by  an  ion  of  greater  potential  energy,  the  proteid  will 
tend  to  be  precipitated,  since  the  negative  part  will  be  partially  neu- 
tralized; and  similarly  if  chlorine  is  replaced  by  an  anion  of  greater 
potential.  Both  ions,  therefore,  will  exert  an  action  in  the  same  direc- 
tion, and  there  will  be  a  summation  of  action  in  this  case  instead  of  a 
difference.     The  formula  for  toxicity  would  become : 

1  Otal  action  =  (^Ication  salt      -^cation  colloids   i  (.-C/anion  salt      -^anion  colloid/ 
^  ^-^cation  salt  '  -^anion  salt/      V-^cation  colloid  "i  -^anion  colloid/   ; 

or,  writing  £'  and  Ea  for  the  ionic  potentials  of  the  anion  and  cation 
of  the  salt  introduced,  and  Ec  and  Ea  for  the  ionic  potentials  of  the 
ions  bound  to  the  colloid, 

log  V'  =  K[{Ei+K)-(Ec+Ej]  +  C 
log  F"  =  i^[(£^+£") -(£.  +  £.)] +  C 


(4) 


Pharmacodynamics  of  Salts  and  Drugs  105 

\ogK.  =  K{E},+K-E}!-I^)  (5) 

TilogT^i^^  •  (6) 


That  is,  the  logarithm  0}  the  ratios  0}  the  dilution  of  the  minimum 
jatal  doses  0}  two  salts,  divided  by  the  difference  oj  the  sums  of  the  ionic 
potentials  oj  the  two  salts,  is  a  constant. 

This  formula  is  very  similar  to  that  derived  by  me  empirically 
from  a  study  of  the  dilutions  of  the  minimum  fatal  doses  of  salts 
toward  the  eggs  of  Fundulus  heteroclitus.     The  empirical  formula  was 

V„ 


Va- 


Ea—  Eo 
2o.  15  +  0.02  Ea 


In  this  formula  Ea  and  Eg  were  the  decomposition  tensions  of  the 
salts. 

If  we  take  instead  of  2  the  base  of  the  Naperian  logarithms  2.718, 

and  instead  of  ; ^  we  write  K,  this  goes  over  into  the  form 

0.l5  +  0.02£a  '  ° 

Taking  natural  logarithms 

log  F<,=log  Vo-K{E,-Eo) 

\og^^=-K{Ea-Eo)  . 

'  o 

This,  in  other  words,  is  the  same  expression  as  that  already  derived, 
using  the  decomposition  tension;  i.  e.,  the  sum  of  the  solution  tensions 
of  the  ions,  in  place  of  the  sum  of  the  ionic  potentials. 

The  formula  may  also  be  derived  in  another  way.  If  V  is  the 
dilution  of  the  minimum  fatal  dose,  and  if  we  let  X  represent  the 
difference  between  the  sum  of  the  ionic  potentials  of  protoplasmic 

dv 
ions  and  salt  ions,  obviously  from  the  form  of  the  curve  -3-  varies 

with  its  f)osition  on  the  curve,  that  is  with  V 

dx  ' 

.-.  log  V=KX  +  C=KiE^  +B-E-E)+C  . 

An  application  of  this  formula  to  the  results  of  McGuigan  and 
myself  give  the  following  values  for  K.     In  each  case  it  is  assumed 


io6 


A.  P.  Mathews 


that    the    original   ions   in    combination   with    the    colloids   are    K 
and  CI.* 

TABLE  5. 


Salts  Compared 


CuCU  -MnCl, 

HgCl,  -MnCU 

NiCh  -MnCh 
AgNOj-HCl.. 

CuCU  -CdCl,. 

NiCl,  -CdCl, 

CdCU  -MgCl, 

HgCh  -MgCh 

CuCl,  -MgCl, 

ZnCl,  -MnCl, 

ZnCl,  -NiCU. 

HgCl,  -CoCh 

HgCU  -CuCh 

CoCh  -MnCU 


e'  +  e'  -e!'-e" 


(1.403) 

(1.816)  - 

(0.848)  - 

(0.8859)' 

(0.7567)" 

(0.201) 

(1-073) 

(2.241) 

(1.828)  ■ 

(0.302) 

(0.546) 

(0.972) 

(0.4121)" 

(0.843  )■ 


log 


3  1249 

3.6812 

2.163 

2 . 0044 

1.765s 

0.8035 

2.1SS3 

4.477 

3 ■ 9208 

1.0430 

1. 1 201 

2.4771 

0.5563 

I .2041 


K 


2.23 
2.03 

2.5s 
2.  26 

2  33 
3-997 
2.010 
1.998 
2.I4S 

3  454 
2.051 
2.548 
I  35 
1.428 


Mean  value  of  .K  2 .  23 

The  values  of  K  (Table  5)  are  on  the  whole  fairly  constant  for  the 
great  majority  of  the  salts  compared.  The  variations  from  the  mean 
of  2 .  2  are  due  almost  entirely  to  the  fact  that  mercury  is  not  so  poison- 
ous as  it  should  be,  and  that  cobalt  is  a  good  deal  less  toxic  than  the 
theory  demands,  while  nickel  is  a  little  more  toxic.  The  explanation 
of  these  variations  is  no  doubt  to  be  found  in  part  in  the  dissociation. 
I  have  assumed  throughout  that  the  dissociation  is  complete.  This 
has  been  done  for  the  sake  of  simpHcity.  It  is,  however,  certain  that 
the  dissociation  of  mercury  chloride  even  in  these  dilutions  is  far  from 

*  Some  modification  or  explanation  is  necessary  of  the  conclusion  of  a  former  paper  that  oppositely 
charged  ions  must  have  of  necessity  an  opposite  action.  This  is  in  one  sense  true.  That  is,  the  {wsitive 
ion  in  combination  with  the  proteid,  if  the  latter  is  electronegative,  must  constantly  be  neutralizing  the 
negative  charge  and  producing  undissociated  albumin.  It  may  be  stated  in  this  sense  that  the  positive 
ion  always  tends  to  precipitate  an  electronegative  albumin.  It  happens,  however,  that  the  power  of 
neutralizing  the  charges  of  the  colloid — that  is,  of  reducing  ionization — varies  greatly  in  different  cations, 
being  greatest  in  those  of  high  ionic  potential,  and  least  in  those  of  low.  If,  therefore,  we  have,  as  we 
do  have  in  a  protoplasmic  system,  colloids  in  a  state  of  equilibrium  with  ions  already  present,  the  particular 
direction  of  the  change  in  state  of  that  equilibrium  produced  by  the  substitution  of  new  positive  or  neg- 
ative ions  for  those  already  present  will  depend  on  the  relative  potentials  of  the  ions  present  and  those 
introduced  in  their  places.  The  actual  effect  observed,  therefore,  of  replacing  an  ion  of  high  potential 
with  that  of  a  low,  will  be  the  direct  opposite  of  that  produced  by  replacing  the  ion  with  an  ion  of  still  higher 
potential,  and  in  this  case  there  will  appear  to  be  an  antitoxic  or  antagonisUc  action  between  two  ions 
of  the  same  character  of  charge.  In  an  earUer  discussion  of  this  matter  I  neglected  to  take  into  account 
the  great  importance  of  the  ions  present  in  protoplasm.  For  example,  suppose  the  ions  in  the  protoplasmic 
system  to  be  mainly  sodium;  and  let  us  suppose  that  potassium  has  a  lower  potential  than  sodium,  while 
calcium  has  a  higher  potential.  If  one  substitute  calcium  for  the  sodium,  the  result  will  be  to  precip- 
itate in  part  the  electronegative  colloids  in  the  protoplasm.  If,  however,  pwtassium  be  substituted  for 
the  sodium,  the  result  will  be  to  dissolve  still  further  the  colloids.  In  this  case  potassium  and  calcium 
will  appear  to  exert  an  antagonistic  action  toward  each  other.  If,  however,  the  ions  already  in  the  proto- 
plasm are  of  higher  potential  than  calcium,  then  both  potassium  and  calcium  will  produce  the  same  kind 
of  an  action  on  the  protoplasmic  colloids.  The  results  obtained  by  Loeb,  Loeb  and  Giess,  Miss  Moore 
and  myself  on  toxic  and  antitoxic  action  of  salts  thus  have  a  very  simple  explanation. 


Pharmacodynamics  of  Salts  and  Drugs 


107 


complete.  If  we  assume  it  to  be  only  50  per  cent,  it  would  bring  the 
mercury  into  its  proper  position.  Similarly  with  cadmium,  which  is 
a  trifle  too  low  in  its  toxicity,  this  dissociation  is  certainly  incomplete. 
As  regards  cobaltous  chloride,  which  is  noticeably  below  what  it 
should  be,  I  have  no  explanation  to  offer  except  to  point  out  that  it 
occupies  the  same  exceptional  position  toward  some  other  forms  of 
protoplasm.  Possibly  its  power  of  forming  double  compounds  with 
ammonia  and  its  derivatives  may  have  something  to  do  with  its 
anomalous  behavior. 

The  constancy  of  K  must  be  regarded,  I  think,  with  the  exceptions 
just  mentioned,  as  satisfactory,  when  it  is  remembered  that  the  method 
of  determining  the  minimum  fatal  dose — that  is,  by  dilution — does 
not  permit  of  very  accurate  figures. 

C  evaluated  from  these  figures  was —  4.1 11.     (See  Formula  4.) 


TABLE  6. 
Resui.ts  on  Fundulus  Eggs. 


Salts  Compared 


CuCh-MnCU 
HgCl,-MnCl, 
HgCl,-CuCU. 
NiCl,-MnCh 
CoCU-MnCl, 
HgCh-MgCl, 
CuCl,-MgCl, 
CuCU-NiCl,  . 
HgCh-CoCl,. 


{e:+e: 

-K-K) 

1  V' 
logp- 

(1.403) 

3S74 

a. 

I 

816 

4 

097 

2 

0 

412 

0 

5229 

I. 

0 

848 

2 

0969 

2 

0 

843 

I 

796 

3 

2 

241 

4 

398 

I . 

I 

828 

3 

87s 

a 

0 

5S6 

I 

477 

a 

0 

972 

2 

30I 

a 

■547 

.  a6o 

.269 

473 

131 

.964 

.  120 

655 

•  367 


Mean  value  K=2.iq 


TABLE  7- 
Comparison  of  Zn  with  all  Other  Metals. 


Salts  Compared 


ZnCl,  ■ 

ZnCl,  ■ 

CdCU  ■ 

CoCU  - 

NiCU  ■ 

PhCU  - 

CuCU  - 

HgCU  • 
AgNOj 


■MgCl, 
-MnCh. 
-ZnCl,. 
■ZnCl,.. 
-ZnClj  . 

ZnCI,.. 
-ZnCU.. 

ZnCl,.. 
-ZnCl.  . 


(£>£:-<-<) 


0.726 
0.302 

0  345 
0.54' 
0.546 
0.613 
1 .  102 

1  514 
1.597 


log 


V. 


602 

301 
194 

5051 
2041 

795 
2  73 
7950 
097 


.581 
.610 
.461 

934 
.374 
.296 
.182 
.186 

3"4 


Mean  K  a .03 

The  results  obtained  upon  Fundulus  (Tables  6  and  7)  do  not  give 
quite  such  constant  values  as  those  of  McGuigan,  and  indeed  with  so 
complex   a   system   this    was   not  to    be    expected.     However,    the 


io8 


A.  P.  Mathews 


average  value  of  K  in  these  results  is  almost  exactly  the  same  as  that 
of  McGuigan;  i.  e.,  about  2.2.  The  exceptions  in  these  results  are, 
with  the  expection  of  cadmium,  the  same  as  those  recorded  by  Mc- 
Guigan, cobalt  being  too  little  toxic  and  zinc  too  toxic.  I  have  deter- 
mined K  for  zinc  chloride  in  comparison  w^ith  all  other  metal  chlorides, 
expecting  that,  while  the  ratio  would  be  too  low  for  all  other  metals 
above  it  in  the  scale  of  ionic  potentials,  it  would  be  too  high  for  all 
metals  below  it,  and  these  two  errors  should  neutralize  each  other, 
provided  the  metals  were  about  equally  distributed  above  and  below 
zinc.  The  result  (Table  7)  gave  for  the  mean  K  2.03,  which  is  a 
litttle  low,  but  fairly  close  to  the  mean  2.2  already  obtained. 

I  have  also  determined  (Table  8)  the  fatal  dose  just  sufficient  to  stop 
swimming  in  two  minutes  in  rapidly  swimming  cultures  of  Volvox 
globator.  Four  metals  were  investigated — i.  e.,  silver,  cadmium, 
manganese,  and  magnesium.  K  was  assumed  to  be  2.2,  the  same  as 
that  for  Fundulus  and  diastase,  and  the  constant  C  was  calculated. 

The  result  was  as  follows : 


TABLE  8. 

Salt 

log  V 

c 

AgNGj 

CdCl, 

MnCU 

MgCU 

5-477 
2.699 
1.699 
0.699 

-3-5 
-3-6 
-3" 
-313 

Mean  — 3.33 


The  constant  C  is  thus  found  as  constant,  as  could  be  expected 
from  the  methods  used  and  the  variability  of  the  cultures. 

Undoubtedly  the  most  consistent  and  accurate  results  thus  far  ob- 
tained are  those  of  McGuigan  upon  the  minimum  fatal  dose  of  salts 
for  the  diastatic  ferment.  These  results  are  plotted  in  Fig.  3.  I  have 
used  only  those  results  which  were  obtained  with  the  metals  Mg,  Mn, 
Zn,  Cd,  Co,  Ni,  H,  Cu,  Hg,  and  Ag,  for  the  reason  that  the  solution  ten- 
sion, and  hence  the  ionic  potential,  of  all  metals  above  magnesium — 
i.  e.,  Ca,  Sr,  Ba,  Na,  K,  Li,  and  Cs —  are  still  so  very  uncertain  as  to 
make  the  comparison  of  ionic  potential  and  fatal  doses  of  little  value. 
In  Fig.  3  the  hne  A  B  represents  the  formula. 

JOg   K  =C- +  A(^£,ca{JQji  salt      -C-cation  colloid j  ~r(.-£^anion  salt      -C-anion  coUoidj  • 


Pharmacodynamics  of  Salts  and  Drugs 


109 


The  ordinates  represent  the  logarithms  (common)  of  the  dilution 
(V)  of  the  minimum  fatal  dose;  the  abscissae  represent  the  differences 
between  the  ionic  potential  of  the  cation  of  the  colloid,  which  is  assum- 
ed to  be  potassium  at  2.9  volts  and  the  ionic  potential  of  the  various 


> 

I 


Mg    2    Mn         Zn 
X= Ionic  Potentials. 


Cd  3  H 
Co 


Cu 


Hg  4  Ac 


Fig.  3.  Plat  of  results  obtained  with  diastase.  Ordinates  are  the  logarithms  of  the  dilution  of  the 
minimum  fatal  dose.  Abscissse  represent  the  difference  between  the  ionic  potentials  of  K..  Cl  and  poten- 
tials of  ions  of  toxic  salts, 

metals.     The  line  makes  an  angle  with  the  A'"-axis,  the  tangent  of 
which  is  2.23,  and  it  cuts  the  F-axis  at  — 4. 11  (or  C). 

The  remarkable  closeness  with  which  the  various  metals  approxi- 
mate to  this  line  will  be  apparent. 


no 


A.  P.  Mathews 


If  now  we  turn  to  my  results  on  Fundulus  (Fig.  4),  two  things  are 
at  once  clear  from  an  inspection  of  Table  6  and  Fig.  4.  The  first 
result  is  that  the  gradient  or  slope  of  the  line  which  represents  the  rela- 
tion between  toxicity  for  Fundulus  eggs  and  the  ionic  potential  is 


Fe"  3  Ni H 
Co  Pb 


Hg  4  AG 


Mg  2         Mn  Zn 

X=Iomc  Potentials. 

Fig.  4.    Plot  of  results  obtained  with  eggs  of  Fundulus  heterodilus.    Ordinates  and  abscissa:  as  in  3. 

exactly  the  same  as  that  found  for  diastase.  This  must  be  regarded 
as  confirmatory  evidence  of  some  value  of  the  probable  correctness 
of  our  attempt  to  work  out  a  numerical  relationship  between 
toxicity  and  ionic  potential.  The  second  result  which  is  very 
clear  is  that  there  is  in  the  case  of  Fundulus  greater  variations  than  in 


Pharmacodynamics  of  Salts  and  Drugs  hi 

the  case  of  the  ferment.  This  is,  of  course,  to  be  expected  since  in  the 
egg  we  are  deahng  with  a  vastly  more  compHcated  system  than  in  the 
ferment.  We  have  not  only  a  variety  of  ferments  and  substances 
which  might  be  differently  affected  by  the  salts,  but  the  eggs  are  in 
addition  separated  from  the  water  in  which  the  salts  are  by  mem- 
branes which  are  known  to  be  variously  permeable  to  different  salts. 
The  fact  that  the  results  show  so  good  an  agreement  with  the  com- 
puted values  is  hence  the  more  satisfactory.  As  regards  the  excep- 
tions, it  will  be  observed  by  comparing  Figs.  3  and  4  that  they  are  in 
general  the  same  in  each,  only  the  deviations  are  greater  for  the  egg. 
Thus  zinc,  which  toward  diastase  was  somewhat  more  poisonous  than 
it  should  be,  toward  the  eggs  is  very  much  more  toxic.  Cobalt,  which 
is  too  little  toxic  toward  diastase,  shows  the  same  relationship  toward 
the  eggs;  the  same  is  true  of  mercury.  On  the  other  hand,  certain 
very  interesting  special  exceptions  occur.  Thus  cadmium,  which 
toward  diastase  occupies  almost  exactly  its  theoretical  position,  is 
toward  Fundiilus  heteroclitus  eggs  extremely  toxic.  It  is,  in  fact,  so 
toxic  and  so  far  out  as  to  show  that  there  is  some  specific  and  special 
reason  for  its  aberrance.  I  have  accordingly  disregarded  it.  On  the 
other  hand,  lead,  which  was  for  some  special  reason  far  out  of  place 
toward  diastase,  is  here  almost  where  it  should  be. 

As  regards  the  toxicity  of  the  metals  sodium,  potassium,  and 
lithium  it  will  be  noticed  that  they  are  relatively  more  toxic  toward 
Fundiilus  than  toward  the  diastase.  The  reason  for  this  may  possibly 
be  that  the  strong  solutions  are  in  themselves  harmful  by  their  osmotic 
action  on  the  cells. 

I  have  also  incorporated  the  results  of  a  few  observations  made 
upon  the  rapidly  swimming  culture  of  Volvox  glohaior  (Fig.  5).  I  de- 
termined the  concentration  just  sufficient  to  stop  swimming  within  two 
minutes.  The  computation  of  the  constant  a,  from  the  results  gives  a 
very  satisfactory  agreement. 

e)  Other  results  on  toxicity. — The  results  of  Caldwell  on  bromelin, 
the  proteolytic  ferment  of  the  pineapple,  I  have  been  unable  to  bring 
to  any  satisfactory  numerical  agreement.  But  while  Caldwell's  re- 
sults cannot  be  brought  into  quantitative  relationship  with  McGuigan's 
and  mine,  the  general  trend  of  the  results  is  plainly  the  same.  The  order 
of  the  toxicity  of  the  metals  is  in  nearly  all  cases  as  it  should  theoreti- 


112 


A.  p.  Mathews 


cally  be,  so  far  as  he  has  tried  those  of  which  the  solution  tension  is 
known.  The  same  exceptions  are  also  apparent.  Thus  cobalt  is 
not  sufficiently  toxic,  and  zinc  is  too  toxic  for  the  rule.  Lead  is  about 
where  it  belongs,  but  in  this  case  barium  is  the  marked  and  peculiar 


> 

BO 
I 


1  Mg  2  Mn  Cd  3  4  Ac 

X=Iomc  Potentials. 
Fig.  5.    Plot  of  results  obtained  with  Volvox.     Abscissae  and  ordinates  as  in  Fig.  3. 

exception,  in  place  of  the  lead  toward  diastase,  and  cadmium  toward 
the  Fundulus  egg. 

The  results  of  Heald  (Table  i)  so  far  they  have  been  obtained 
with  the  salts  we  are  examining,  show  much  the  same  order  of  effi- 
ciency. Thus  for  Pisum  sativum  the  order  of  toxicity  is :  Ag,  Hg, 
Cu,  Ni,  Co,  and  H;  while  for  Zea  mais  it  is  Ag,  Cu,  Hg,  Ni,  Co, 
and  H.  These  results  are  not  of  a  sufficiently  definite  character, 
based  as  they  are  on  the  growth  of  roots,  to  enable  very  accurate  quan- 


Pharmacodynamics  of  Salts  and  Drugs  113 

titative  comparisons.  It  will  be  noticed  that  nickel  is  more  poisonous 
than  cobalt  for  Pisum  sativum,  and  far  more  poisonous  than  cobalt 
for  Zea  mais.  Both  of  these  roots  appear  less  sensitive  to  acids 
than  to  the  metals,  the  hydrogen  ion  being  less  toxic  than  nickel. 
With  Lupinus,  while  the  general  order  is  the  same  as  that  observed 
elsewhere,  cadmium  is  here  more  poisonous  than  copper — just 
the  exception  noted  for  Fundulus. 

The  results  of  Clark  and  Stevens  upon  mold  spores  are  also  for  the 
purposes  of  quantitative  comparisons  unsatisfactory.  These  spores 
appear  to  be  surrounded  by  such  membranes,  or  to  have  so  great 
a  resistance,  as  to  make  the  interpretation  of  results  very  doubtful, 
although  there  can  be  no  doubt  that  the  general  trend  of  the  results 
is  the  same  as  that  already  noted. 

True  and  Kahlenberg's  results  (Table  i)  are  more  satisfactory, 
but,  owing  to  the  fact  that  these  authors  did  not  study  many  of  the 
metals,  and  also  did  not  pretend  to  fix  the  fatal  point  with  accuracy, 
their  results  are  not  satisfactory  for  quantitative  treatment.  True's 
results  upon  the  toxicity  of  the  salts  of  the  various  acids  are  unfortu- 
nately unavailable  for  our  purposes,  owing  to  the  uncertainty  of 
the  ionic  potential  of  these  anions. 

/ )  The  soluhility  of  globulin  in  salt  solutions. — In  a  previous 
paper'  I  showed  that  the  solubility  of  sodium  albuminate  (egg  albu- 
min in  alkaline  solution)  in  different  salt  solutions  was  determined 
by  the  tension-coefficient  of  the  salt.  By  the  tension-coefficient 
was  meant  the  difference  between  the  solution  tensions  of  the  ions 
divided  by  their  sum.  The  numerator  of  this  fraction  should  be 
the  ionic  potentials  instead  of  the  solution  tensions.  I  showed  that 
in  an  alkaline  solution  the  solubility  was  greater  in  sodium  iodide 
than  in  the  bromide  or  chloride,  and  that  when  the  ionic  potential 
of  the  cation  surpassed  a  certain  figure  the  salts  precipitated  the 
albumin.  Table  II  ^  shows  the  relationship  (qualitative)  between 
the  ionic  potentials  of  salts  and  their  power  of  dissolving  or  pre- 
cipitating such  albumin,  both  the  unboiled  and  the  boiled.  It 
will  be  seen  by  an  inspection  of  the  table  that  the  ionic  potential 
arranges  the  salts  in  the  order  of  their  action  on  the  albumin. 

Osborne  and  Harris^  have  estimated  quantitatively  the  solvent 

'Mathews,  Amer.  Jour.  Physiol.,  igos,  14.  p.  204. 
'  Osborne  and  Harris,  ibid.,  igos,  p.  151. 
i  Mathews,  loc.  cil.,  p.  211. 


114 


A.  P.  Mathews 


power  of  many  salts  for  the  globulin  of  the  hemp  seed  edestin.  I 
append  their  results  here  to  show  how  far  they  agree  with  the  theoreti- 
cal deductions  which  were  worked  out  on  p.  102.  In  Table  9  Ec  —  Ea 
represents  the  difference  between  the  ionic  potentials  of  the  salts; 


TABLE  9. 


Salt 

E-E, 

c.c.  to  Dissolve 
igr. 

K 

KI 

-2.123 
-1. 6s 
— 1,226 
-1-743 

-1.27 

-0.846 

—  1. 10 

—  0 . 646 

KBr 

KCl 

Nal 

NaBr 

NaCl 

LiBr 

10 
IS 
5 
9 
12 
12 
19 

I 
7 

I 
I 

■  51 

-44 

•44 
■33 

LiCl 

■  44 

the  third  column  represents  the  number  of  c.c.  of  a  normal  solution 
of  the  salts  which  will  just  dissolve  one  gram  of  edestin.  I  have 
taken  these  figures  from  the  chart  given  by  Osborne  and  Harris.  They 
are  only  approximate.  The  theory  requires  that  the  more  negative  the 
difiference  Ec  —  E^,  the  smaller  the  amount  of  salt  necessary  to  dis- 
solve a  given  amount  of  the  edestin.  It  will  be  seen  that  this  relation- 
ship holds  very  well  if  we  compare  the  iodides,  chlorides,  and  bromides 
of  sodium,  lithium,  and  potassium  respectively.  Unfortunately, 
the  great  uncertainty  of  the  solution  tensions  and  ionic  potentials 
of  sodium,  potassium,  and  lithium,  prevent  quantitative  comparisons 
between  the  different  metals.  In  the  fourth  column  under  K,  I 
have  computed  the  constant  by  the  formula  on  p.  102,  using  instead 
of  the  logarithm  of  the  dissolution  the  logarithm  of  the  ratios  of 
number  of  c.c.  of  the  different  solutions  necessary  to  dissolve  one 
gram.  The  figures  are  placed  between  the  salts  compared.  The 
formula  used  was: 

T  C.C.T 


c.c. 


Osborne  and  Harris  draw  the  conclusion  that  the  solubility 
is  independent  of  the  nature  of  the  base,  but  I  think  their  figures 
speak  for  themselves.  The  differences  between  potassium,  lithium, 
and  sodium  are  small,  to  be  sure,  but  nevertheless  apparent.  The 
importance  of  the  base  becomes  obvious  so  soon  as  any  other  salts 
are  examined  in  which  the  base  has  a  higher  ionic  potential  than 


Pharmacodynamics  of  Salts  and  Drugs  115 

these.  Then  it  is  seen  that  whether  the  sah  dissolves  the  edestin 
or  converts  it  into  a  curdy  mass  depends  mainly  on  the  base.  The 
reason  why  so  slight  differences  exist  in  the  solvent  power  of  the 
cations,  sodium,  potassium,  lithium,  barium,  calcium,  and  magne- 
sium, is  shown  by  an  examination  of  their  ionic  potentials,  which 
are  very  low  and  probably  about  the  same  in  each.  The  solvent 
power  of  manganese  and  ferrous  chlorides  is  low. 

Cu,  Cd,  Cr,  Co,  Fe'",  Pb,  Hg,  Cu,  Al,  Zn  chlorides  and  nitrates 
all  fail  to  dissolve. 

The  solvent  powers  of  the  anions  arrange  themselves  in  descending 
order  as  follows:  CrO^,  SO3,  8^03,  I,  Br,  CI,  SO4,  which  is  almost 
certainly  the  descending  order  of  the  ionic  potential  of  these  ions. 
The  fact  that  these  bivalent  ions  of  high  ionic  potential  dissolve,  in- 
stead of  precipitating,  the  colloid,  and  that  the  valence  of  the  anion 
is  unimportant,  is,  in  my  opinion,  good  evidence  that  the  edestin 
in  such  solutions  is  electronegative  and  not  electropositive;  for,  as 
has  been  shown  by  Hardy  and  many  others,  the  valence  of  the  ion 
of  the  same  sign  as  the  colloid  is  immaterial,  but  toward  a  colloid  of 
opposite  sign  it  is  very  important. 

The  peculiarity  of  the  dissolving  action  of  the  heavy  metal  ace- 
tates' also  receives  a  possible  explanation.  In  these  solutions  which 
dissociate  acetic  acid  the  edestin  becomes  electropositive.  Con- 
sequently it  is  no  longer  precipitated  by  the  positive  ions,  but  receives 
energy  from  them,  and  is  rendered  more  positive,  and  hence  more 
soluble,  than  it  was  before.  The  cause  of  the  failure  of  the  chlorides 
to  dissolve  the  edestin  in  acid  solution  would  be  that  given  by 
Osborne,  that  in  such  solutions,  where  there  are  many  hydrogen 
ions,  it  is  changed  into  edestan  as  an  insoluble  product. 

I  append  here  also  a  summary  of  the  results  of  Pauli^  illustrating 
the  same  facts,  showing  the  parallelism  between  the  solvent  or  pre- 
cipitating power  of  the  anion  upon  albumin  and  its  potential  energy 
content.     The  parallelism  is  certainly  unmistakable. 

Order  of  precipitation SCN>I>Br>NO.,>Cl>C,H,Oj 

Ionic  potential 83(  ?)  —  . 79- 1.27— 1.694 

g)  The  phenomena  0}  stimulation  of  cells. — I  found  that  the  salts 
ranged  themselves  very  simply  in  the  order  of  their  encrg}'  content, 

'Osborne  and  Harris,  loc.  cit.,  p.  165. 

'  Pauli,   Hojmeister's  BeUrdge,   1905,   6,   p.   249. 


ii6  A.  P.  Mathews 

so  far  as  their  action,  stimulation  or  depression,  on  the  motor  nerve 
was  concerned.  But  it  is  clear  from  what  has  been  said,  that  no 
such  simple  arrangement  is  to  be  anticipated  in  studying  a  complex 
system  such  as  a  cell  undergoing  rapid  change.  In  such  a  system 
all  we  observe  from  the  action  of  the  salt  is  a  definite  result,  which 
implies  a  certain  change  in  the  system.  This  result  may  be  a  stimula- 
tion, such  as  a  muscle  contraction,  or  nerve  impulse.  Evidently 
the  same  result  may  be  brought  about  in  several  different  ways: 
either  by  direct  action  of  the  salt  on  the  particular  part  of  the  system 
which  undergoes  change ;  or,  indirectly,  by  the  salt  altering  another 
part  of  the  system,  so  as  to  produce  or  check  the  result.  It  is  con- 
ceivable that  the  same  salt  may  have  a  double  action :  by  one  action 
tending  to  produce  the  direct  change  of  the  response ;  and  indirectly 
by  action  on  another  part  of  the  system  having  as  a  result  the 
setting-up  a  process  which  will  check  its  own  direct  action. 

Something  of  this  last  process  obtains,  I  believe,  in  protoplasm 
generally,  so  that  strict  adherence  to  the  law  of  ionic  potential  action 
is  not  to  be  expected;  but  a  general  adherence  is  to  be  expected, 
and  the  facts  show  it  exists. 

An  interesting  example  of  this  stimulating  action  is  seen  in 
the  extrusion  of  polar  globules  in  Chaetopterus  eggs.  These  eggs 
undergo  the  first  processes  of  maturation  before  they  are  shed,  but 
they  do  not  extrude  the  polar  globules  until  fertilized.  The  first 
polar  spindle  is  formed  and  comes  to  rest  in  the  equatorial  plate- 
stage.  Evidently  in  this  egg  there  is  not  sufficient  energy  in  the 
spindle  to  overcome  the  resistance  offered  by  the  surface  tension  or 

TABLE  lo. 

MiNiMUN  Concentration  of  Various  Salts  Causing  Extrusion  of  Polar 
Globules  in  Chaetopterus. 

Salt  Concentration 

Na3  citrate jjg  n 

Na,S04 \   n 

KI <^^n 

KBr <^  n 

KCl ^s  n 

NaCl /j  « 

LiCl <i« 

CaClj tV  '^ 

MgCl. o(?) 

MnCU o 

CdCU <i;5wr« 

^^^'2 <  24,500^ 


Pharmacodynamics  of  Salts  and  Drugs  i  i  7 

other  factors,  of  the  egg.  Sahs  may  cause  extrusion  either  by 
increasing  the  energy  in  the  egg,  or  by  decreasing  the  surface  tension. 
Unfortunately  the  end-points  were  not  determined  accurately  in 
many  instances,  but  the  figures  (Table  10)  suffice  to  show  that  the 
stimulation  of  this  particular  function  was  greatest  in  copper  and 
cadmium,  fell  to  nothing  in  manganese,  was  doubtful  in  magnesium, 
and  then  increased  as  salts  having  anions  of  higher  ionic  potential 
were  used.  In  other  words,  if  we  begin  with  manganese  or  magne- 
sium chlorides  and  pass  to  salts  having  a  higher  potential  energy  of 
either  the  anion  or  cation,  this  function  is  stimulated,  and  less  of  the 
salt  must  be  used  as  the  energy  content  increases. 

GENERAL  CONCLUSONS. 

The  general  conclusions  of  this  investigation  are: 

1.  The  action  of  salts  upon  the  protoplasmic  system  is  due  chiefly 
to  the  ions  of  the  salt. 

2.  The  particular  result  obtained — toxic,  stimulation,  or  depres- 
sion excited  by  any  salt  solution — is  caused,  in  part  at  least,  by  the 
substitution  of  the  ions  in  the  protoplasmic  system,  and  in  large  measure 
in  combination  with  the  protoplasmic  colloids,  by  the  ions  of  the 
salt  solution  used, 

3.  This  substitution  causes  a  disturbance  of  the  equilibrium 
of  the  protoplasmic  system,  which,  if  sufficiently  pronounced,  leads 
to  destruction. 

4.  The  power  0}  different  ions  to  upset  the  ordinary  state  of  the 
protoplasmic  system  depends  on  the  difference  between  the  potential 
energy  content  0}  the  ion  which  is  replaced,  and  that  which  is  intro- 
duced. This  difference  in  potential  energy  content  is  determined  by 
the  difference  in  the  intensity  factor  of  the  potential  energy  of  the 
ions — i.  e.,  by  differences  in  the  ionic  potentials. 

5.  It  follows  from  4  that  the  ions  must  arrange  themselves  in 
toxic  power  according  to  their  available  potential  energ)'  contents 
(ionic  potentials).  This  was  shown  to  be  the  case  not  only  for  toxic 
action,  but  also  for  stimulating  and  depressing  action. 

6.  It  was  shown  that  a  good  numerical  relation  exists  between 
the  available  potential  energ}'  of  any  salt  and  its  minimum  fatal 
dose,  so  that  for  simple  systems  the  minimum  fatal  dose  can  be  very 


ii8  A.  P.  Mathews 

closely  calculated  from  the  available  potential  energy  of  the  salt, 
if  certain  constants  are  known. 

7.  A  method  was  found  for  computing  the  ionic  potential  of 
various  ions  from  the  solution  tensions. 

8.  In  the  case  of  Fundulus  eggs,  which  were  particularly  inves- 
tigated from  this  point  of  view,  the  order  is  also  consonant  with 
the  theory,  but  the  quantitative  relationships,  while  fairly  good, 
are  not  so  uniform  as  in  the  case  of  diastase. 

The  character  of  the  action  of  any  given  salt  solution  on  proto- 
plasm must  of  necessity  depend  upon  the  character  of  the  ions,  metal 
and  metalloid,  already  in  combination  with  the  protoplasm.  This 
results  as  a  necessary  consequence  of  the  theory,  and  it  explains 
the  fact  that  toward  different  cells,  and  toward  the  same  cell  after 
exposure  to  different  salt  solutions,  the  same  salt  solution  may  exert 
a  different  action,  being  at  times  stimulating,  at  other  times 
depressing. 

It  has  been  shown  that  the  physiological  action  of  any  salt  solu- 
tion is  a  function  of  the  available  potential  energy  of  its  ions.  The 
action  of  the  organic  drugs  will,  I  think,  also  be  found  to  depend, 
in  part  at  least,  on  the  available  potential  energy  of  the  dissociated 
particles  of  the  drug. 


AN  INSTANCE  OF  THE  APPARENT  ANTITOXIC  ACTION 

OF  SALTS  * 

Percy  Goldthwait  Stiles  and  Carl  Spencer  Millie  en. 

(From  the  Physiological  Laboratory  of  the  Massachusetts  Institute  of  Technology  and  the  Biological  Depart- 

ment   of  Ripon   College.) 

The  idea  that  certain  salts  may  neutralize  toxic  properties  of 
other  salts  so  that  a  suitably  proportioned  mixture  may  be  harmless 
to  living  protoplasm  though  its  constituents  are  individually  harmful, 
is  one  which  we  owe  especially  to  Loeb'  and  Mathews.*  There 
is  much  evidence  favoring  such  a  conception.  The  observations 
of  Loeb  upon  the  abnormal  behavior  of  skeletal  muscles  in  sodium- 
chloride  solutions  and  upon  the  failure  of  fish  embryos  to  develop 
in  solutions  of  this  single  salt  are  likely  to  be  ranked  as  classic.  He 
found  that  the  muscles  no  longer  twitched  when  certain  salts,  notably 
those  of  calcium  and  potassium,  had  been  added  to  the  bath,  and 
that  the  embryos  prospered  when  similar  corrective  additions  to 
the  medium  had  been  made. 

These  instances  no  longer  stand  alone,  for  they  have  been  repeat- 
edly paralleled  ;3  but  the  cases  first  described  remain  fair  types 
of  the  class.  Whether  we  do  well  to  use  the  terms  "toxic"  and 
"antitoxic"  in  this  connection  may  be  questioned,  and  depends 
upon  what  theory  we  adopt  to  account  for  the  facts.  The  termi- 
nology has  been  criticised  by  Howell,-*  and  recently  by  Osborne.'' 
When  it  is  said  that  a  certain  salt  by  itself  is  toxic,  the  meaning 
seems  often  to  be  merely  that  it  is  insufficient,  and  to  say  that  a 
second  salt  is  antitoxic  to  the  first  implies  little  more  than  the  thought 
that  it  supplies  some  need  of  the  tissue  not  met  by  the  other.  But 
we  may  keep  the  picturesque  and  convenient  expressions  without 
being  dogmatic  as  regards  their  application. 

In  a  recent  paper*^  the  authors  dealt  with  the  action  of  lithium 
upon  skeletal  muscle.  The  experiments  recorded  at  that  time  gave 
ground  for  the  belief  that  lithium-chloride  solutions  are  among 
the  least  harmful  to  which  the  muscle  may  be  exposed,  and  that 
such  solutions  preserve  the  tissue  very  nearly  as  well  as  the  usual 

♦Received  for  publication  December  14,  1905. 

119 


I20  P.  G.  Stiles  and  C.  S.  Milliken 

"physiological"  mixtures.  At  the  conclusion  of  that  paper  we 
questioned  whether  it  might  not  be  possible  to  find  a  mixture  of 
lithium  chloride  with  some  other  salt  which  should  still  more  closely 
approach  the  normal  fluid  in  its  conserving  properties.  When 
we  expressed  this  hope,  we  had  already  made  some  tentative  experi- 
ments with  suggestive  results.  These  we  have  since  repeated  and 
extended,  and  our  observations  may  now  be  outlined. 

Some  time  ago  we  investigated  the  duration  of  irritability  and 
the  working  capacity  of  muscles  immersed  in  solutions  of  magne- 
sium chloride.  We  found  that  the  loss  of  excitabihty  is  rapid, 
and  that  two  or  three  hours  in  such  a  bath  makes  the  gastrocne- 
mius unresponsive  to  the  strongest  stimulation.  Thus  solutions  of 
magnesium  chloride  alone  are  distinctly  unfavorable  to  the  muscle 
protoplasm.  However,  we  were  led  to  test  mixtures  of  the  chlo- 
rides of  lithium  and  magnesium  (with  the  former  in  large  excess), 
and  to  compare  the  records  written  by  muscles  treated  with  these 
mixtures  with  those  obtained  from  companion  muscles  in  lithium 
chloride  alone.  It  seemed  not  unlikely  that  these  combinations 
might  prove  somewhat  superior  to  the  straight  hthium  chloride, 
as  well  as  vastly  better  than  the  unmixed  magnesium  chloride.  In 
the  current  phraseology  we  looked  for  an  antitoxic  action  on  the 
part  of  magnesium. 

The  specific  influence  of  magnesium  salts  upon  various  living 
tissues  has  not  been  sufficiently  investigated.  At  the  time  of  this 
writing*  there  has  appeared  the  first  of  a  promised  series  of  papers 
by  Meltzer  and  Auer,^  which  will  doubtless  make  much  new  and 
exact  information  available.  As  these  writers  point  out,  the  con- 
spicuous property  of  magnesium  salts  in  large  quantity  is  a  depres- 
sing or  paralyzing  one,  and  they  suggest  that  the  physiological  role 
of  these  salts,  which  are  somewhat  abundant  in  animal  cells  and 
fluids,  is  inhibitory.  If  this  is  the  case,  there  is  a  closer  relation- 
ship between  magnesium  and  potassium  than  between  magnesium 
and  calcium.  It  should  be  easy  to  define  the  comparative  effects 
and  the  antagonisms  of  these  salts  by  experiments  with  plain  and 
cardiac  muscle,  and  we  have  undertaken  work  along  these  lines. 

In  the  present   contribution   we  shall   confine  ourselves  to  the 

♦November,  1905. 


Apparent  Antitoxic  Action  of  Salts  121 

effect  upon  skeletal  muscle  of  various  mixtures  in  which  the  chlo- 
rides of  lithium  and  magnesium  were  the  principal  ingredients.* 

When  a  muscle  is  exposed  to  a  bath,  there  are  at  least  three  ways 
in  which  we  may  estimate  the  effects  of  the  immersion.  First, 
we  may  simply  find  out  the  duration  of  its  irritability,  noting  when 
in  the  course  of  hours  or  days  it  ceases  to  respond  to  strong  stimuli ; 
in  this  case  we  should  apply  shocks  only  occasionally,  and  the  muscle 
would  not  have  performed  much  work.  Second,  we  may  exhaust 
the  muscle  by  severe  and  repeated  stimulation  after  a  longer  or 
shorter  period  in  the  bath,  and  thus  obtain  a  rough  measure  of  its 
working  capacity.  For  this  purpose  a  work-adder  may  be  used, 
or  the  area  of  the  record  traced  on  a  slowly  moving  surface  may 
be  noted.  Third,  we  may  determine  the  irritability  of  the  immersed 
muscle  from  time  to  time  by  varying  the  strength  of  the  stimuli 
and  recording  the  position  of  the  secondary  coil  corresponding 
to  maximal  and  minimal  responses.  We  kept  all  three  points  in 
view,  but  placed  emphasis  upon  the  second — the  total  performance 
of  the  muscles  when  stimulated  strongly  and  frequently.  Some- 
times a  thousand  contractions  went  to  form  a  record. 

All  our  experiments  were  comparative.  We  used  companion 
muscles  from  the  right  and  left  legs  of  frogs,  so  that  we  might  have 
controls  which  at  the  outset  should  be  practically  identical  with 
their  fellows  in  potential  energy  and  irritability.  The  muscles 
worked  upon  equal  levers  similarly  adjusted  and  weighted.  The 
stimulating  current  passed  through  both  preparations,  traversing 
each  lengthwise  and  being  led  from  one  to  the  other  by  a  flexible 
bit  of  metallic  tinsel.  In  most  of  the  experiments  the  shocks  were 
derived  from  a  cog-wheel  interrupter  driven  by  clock-work.  Break- 
shocks  alone  were  brought  to  bear  on  the  tissue,  the  makes  being 
eliminated  by  a  mechanical  device.  The  stimuli  were  applied 
from  30  to  60  times  per  minute.  Usually  the  stimulation  was  supra- 
maximal, and  the  muscles  were  out  of  the  solutions  when  working, 
so  that  there  could  be  no  scattering  of  the  current.  They  were 
frequently  washed  during  their  prolonged  series  of  contraction 
The  solutions  were  made  from  salts  of  high  purity  and  tested  as 

♦Sometimes  the  chlorides  of  caldum  and  potassium  were  also  present  in  the  small  percentages  of  a 
Ringer's  mixture.  Their  presence  did  not  affect  the  general  nature  of  the  results,  and  it  is  needless  to 
dwell  upon  the  minor  modifications  which  they  probably  brought  about. 


122  P.  G.  Stiles  and  C.  S.  Milliken 

to  the  freezing-point.  By  this  means  we  were  able  to  use  media 
which  had  a  uniform  osmotic  pressure  appropriate  to  the  frog's 
tissues.     The  value  A  varied  little  from  o .  50°. 

About  thirty  experiments  of  a  reasonably  consonant  character 
yield  data  on  which  the  following  conclusions  are  based.  The 
order  and  duration  of  the  different  trials  were  purposely  contrasted 
as  far  as  possible.  Sometimes  the  immersions  were  measured  in 
minutes,  sometimes  in  days.  When  low  temperatures  were  main- 
tained, it  was  found  possible  to  keep  preparations  for  nearly  a  week 
in  extreme  instances. 

Skeletal  muscles  are  better  preserved  by  a  proper  mixture  of 
lithium  and  magnesium  chlorides  than  by  lithium  chloride  alone. 
It  is  naturally  difRcult  to  fix  upon  the  optimum  ratio  between  the 
two  chlorides.  Seven  parts  by  volume  of  lithium-chloride  solution 
to  one  part  of  magnesium  chloride  is  a  highly  favorable  propor- 
tion. If  we  increase  the  magnesium  much  above  this  fraction, 
it  shows  its  influence  in  lowering  the  irritabihty  of  the  muscle. 

The  mixtures  favor  a  maximal  performance  of  work  by  muscles 
whether  the  exposure  has  been  short  or  long.  This  is  apparently 
not  true  unless  the  stimulation  is  amply  strong,  for  the  irritability 
of  the  muscle  is  probably  somewhat  depressed  in  the  presence  of 
even  a  small  quantity  of  magnesium  salts.  Closely  connected 
with  this  is  the  fact  that  the  earlier  contractions  of  the  muscle  in 
straight  lithium  chloride  may  exceed  those  of  the  companion  in 
lithium-magnesium  mixture,  yet  the  latter  outdoes  the  former  be- 
cause it  outlasts  it.  This  seems  to  us  a  very  significant  fact;  per- 
haps we  have  here  the  key  to  the  beneficial  working  of  magnesium. 
It  may  be  conceived  to  have  the  action  of  economizing  the 
metabohsm,  of  preventing  a  wasteful  expenditure  of  substance 
and  energy  in  the  muscle-cells.  We  have  noticed  that  it  lessens 
contracture  in  fatigued  muscles.  This  is  thoroughly  in  harmony 
with  the  broad  statements  of  Meltzer  and  Auer,  whose  work  was  not 
known  to  us  when  we  were  first  led  to  these  opinions. 

We  believe,  then,  that  magnesium  in  small  quantities  is  a  con- 
serving element  in  contractile  tissue.  We  may  again  question 
whether  it  is  best  to  designate  this  action  as  antitoxic,  though  there 
is  a  marked  similarity  between  our  experiments  and  those  upon 


Apparent  Antitoxic  Action  of  Salts  123 

eggs  and  larvae,  in  which  the  antagonism  of  ions  has  been  made 
apparent.  It  is  not  necessary  to  assume  that  there  is  any  pecuhar 
power  of  mutual  neutralization  in  lithium  and  magnesium.  We 
may  reasonably  suppose  that  the  plain  lithium-chloride  bath  is 
not  of  the  most  favorable  nature,  because  the  metabolism  of  the 
muscle  immersed  in  it  is  not  of  the  most  economical  character. 
An  admixture  of  magnesium  chloride  secures  economy  and  a  better 
direction  of  the  energy  set  free.  But  an  excess  of  magnesium  chlo- 
ride is  naturally  unfavorable,  because  the  same  action  carried  farther 
must  result  in  depressed  excitabihty. 

It  seems  to  us  a  legitimate  expectation  that  many  other  cases 
in  which  mixtures  of  two  salts  have  proved  superior  to  either  by 
itself  may  be  explained  in  equally  simple  ways. 

REFERENCES. 

1.  LoEB.     Festschr.  f.  Fick,  Braunschweig,  1899,  p.  101;  Amer.  Jour.  Physiol.,  1900, 
pp.  327,  383,  434;  ibid.,   1902,  6,  p.  411;  Arch  j.  ges.  Physiol.,  1901,    88,  p.  68. 

2.  Mathews.     Amer.  Jour.  Physiol.,  1904,  10,  p.  290;  ibid.,  1905,  12,  p.  419. 

3.  Moore.     Amer.  Jour.  Physiol.,   1902,   7,  p.   i. 

LiLLiE.     Ibid.,  1901,  5,  p.  56;  ibid.,  1902,  7,  p.  25;  ibid.,  1904,  10,  p.  419. 
Neilson.     Ibid.,   1902,   7,  p.   405. 
McGuiGAN.     Ibid.,  1904,  10,  p.  444. 
Maxwell.     Ibid.,  1904,  13,  p.  154. 
Benedict.     Ibid.,   1905,   13,  p.    192. 

4.  Howell.     Amer.   Jour.   Physiol.,    1901,   6,   p.    181. 

5.  Osborne.     Jour.   Physiol.,    1905,   33,   No.    i,   p.    10. 

6.  MiLLlKEN  AND  Stiles.     Amer.  Jour.  Physiol.,   1905,   14,  p.  359. 
7    Meltzer  and  Auer.     Ibid.,  1905,   14,  p.  366. 


EXPERIMENTS  WITH  BACTERIAL  ENZYMES  * 

Edwin  O.  Jordan. 

Introductory. 

Technique. 

Conditions  of  Gelatinase  Formation. 

Composition  of  Medium. 

Reaction  of  Medium. 
Physical  Characters. 

Resistance  of  Gelatinase  to  Heat. 

Filtration. 
Conditions  of  Activity. 

Reaction  of  Medium. 

Temperature. 
The  Action  of  Formalin  upon  Liquefied  Gelatin. 

The  Relation  Between  Bacterial  Gelatinases  and  Bacterial  Hemolysins. 
Summary  and  Conclusions. 

INTRODUCTORY. 

It  was  first  shown  in  1886  by  Bitter'  that  the  liquefaction  of  gelatin 
by  bacteria  depends  upon  enzyme  action.  Rietsch,^  Senger,^ 
Jerosch/  Sternberg,  ^  Krabbe,^  and  others  confirmed  and  somewhat 
extended  Bitter's  observations,  and  in  1890-92  Fermi^  published 
comprehensive  investigations  upon  the  properties  of  gelatin-liquefying 
and  other  bacterial  enzymes. 

The  power  of  bacterial  filtrates  to  liquefy  gelatin  is  a  more  or  less 
independent  quality.  It  is  true  that  various  experimenters  have  not 
distinguished  between  the  gelatin-liquefying  ferments  and  other  "pro- 
teolytic" enzymes,  and  have  openly  or  tacitly  assumed  that  gelatino- 
lytic  ability  is  a  measure  of  general  proteolytic  power.  Malfitano,* 
however,  has  shown  that  the  albuminolytic  and  gelatinolytic  proper- 
ties of  anthrax  filtrates  are  really  distinct  and  should  not  be  con- 
founded. This  writer  states  that  it  is  possible  to  weaken  or  even 
destroy  the  albuminolytic   power  of    an    anthrax   filtrate   without 

*  Received  for  publication  January  3,  igo6. 

'  Arch.  j.  Hyg.,  1886,  5,  p.  245.  *  Baumgarten's  Jahrb.,  1887,  3,  p.  104. 

•  Jour,  de  pharm.  et  de  chimie,  1887,  16,  p.  8.  '  Ibid,  p.  363. 

*  Baumgarten's  Jahrb.,  1887,  3,  p.  104.  *  Jahrb.  wiss.  Bot.,  1890,  21,  p.  32°- 
'  Centralbl.  /.  Bakl.,  1890,  7,  p.  469;  ibid.,  1891,  10,  p.  401;  ibid.,  1892,  12,  p.  713. 

•  Compt.  rend,  de  la  Soc.  de  Biol.,   1903,  55,  p.  843. 

124 


Experiments  with  Bacterial  Enzymes  125 

materially  attenuating  its  gelatinolytic  power.  As  a  matter  of  fact, 
Rietsch'  in  1887  called  attention  to  the  existence  of  at  least  two 
dfferent  proteolytic  ferments,  and  Fermi^  in  1891  pointed  out  that 
the  action  of  the  bacterial  ferments  upon  fibrin  and  egg  albumin  was 
relatively  feeble  and  was  not  correlated  with  their  action  upon  gela- 
tin.3  PoUak  has  recently  stated  that  serum  digestion  and  gelatin 
digestion  by  trypsin  solutions  depend  upon  the  action  of  two  different 
ferments.  There  is  reason,  therefore,  for  considering  the  gelatinases"* 
as  a  definite  class  of  bacterial  proteases.  According  to  Mavrojannis,^ 
two  kinds  of  gelatinases  occur:  (i)  those  that  decompose  gelatin 
with  the  formation  of  gelatoses,  and  (2)  those  that  push  decomposi- 
tion as  far  as  the  formation  of  gelatin  peptones  and  perhaps  beyond. 
As  will  be  shown  presently,  however,  it  is  doubtful  if  this  distinction 
can  be  maintained. 

TECHNIQUE. 

In  the  work  here  described,  a  uniform  procedure  has  been  followed 
in  testing  the  gelatinolytic  action  of  bacterial  filtrates  and  cultures. 
Five  c.c.  of  neutral  carbol  gelatin  (8.0  per  cent  gelatin;  o. 25  per  cent 
phenol)  has  been  used  as  the  standard  quantity  to  be  subjected  to 
enzymic  action;  a  measured  quantity  of  the  enzyme-containing  fluid 
is  added  to  this  while  the  gelatin  is  warm  (35°-4o°) ;  the  control  tubes 
are  diluted  with  0.85  per  cent  NaCl  solution  correspondingly;  en- 
zyme and  gelatin  are  thoroughly  mixed  by  shaking,  and  incubation  at 
36°-37°  for  20  hours  follows.  The  tubes  are  then  cooled  in  ice- water 
for  1 5  minutes,  after  which  they  are  removed  and  allowed  to  stand  at 
room  temperature  for  15  minutes,  when  the  extent  of  solidification  is 
recorded.  In  case  the  action  of  the  enzyme  is  complete,  the  contents 
of  each  tube  remain  entirely  liquid.  I  have  termed  the  smallest 
amount  necessary  to  produce  complete  liquefaction  under  the  condi- 
tions specified  the  minimum  lytic  dose.  If  the  action  is  only  partial 
the  lower  half  or  two-thirds  of  the  gelatin  may  solidify,  or  the  whole 
mass  may  be  semi-solid.  Control  tubes  incubated  for  a  similar 
period    show  complete   solidification  without   a  trace  of  tremor  on 

■  Loc.  cil.,  p.  II. 

'  Cenlralbl.  f.  Bakl.,  i8gi,   lo,  p.  401. 
J  Beitrdge  z.  cliem.  Physiol,  u.  Pathol.,   1904,  6,  p.  95. 
«  PoUak  uses  the  term  "glutinases  ." 
s  Ztschr.  /.  Hyg.,  1903,  45,  p.  108;  Compt.  rend,  de  la  Soc.  de  Biol.  iqo»,  55,  p.  1605. 


126  Edwin  O.  Jordan 

shaking.*     The  reactions  given  for  the  nutrient  media  are  based  on 
the  phenolphthalein  neutral  point. 

CONDITIONS    OF    GELATINASE   FORMATION. 

Composition  of  medium. — One  inquiry  of  obvious  theoretical 
interest  is  whether  gelatinase  is  formed  more  abundantly  in  gelatin- 
containing  media  than  in  media  in  which  no  gelatin  is  present. 
Lauder  Brunton  and  Macfadyen/  as  the  result  of  their  studies,  con- 
cluded that  "an  enzyme  is  formed  in  meat  broth  which  liquefies  gela- 
tin, and  does  so  more  surely  and  quickly  than  the  enzyme  formed  in 
gelatin  itself."  Fermi/  on  the  other  hand,  found  that  in  general  the 
enzyme  production  in  broth  was  less  abundant  than  in  nutrient  gela- 
tin. Recently  Abbott  and  Gildersleeve^  have  stated  that  in  their 
observations  "the  enzyme  content  of  completely  liquefied  gelatin  cul- 
tures was  always  marked,  and  was  in  general  more  marked  for  all 
species  than  was  the  case  with  any  of  the  other  culture  media  used." 
These  authors  attach  importance  to  this  finding  as  tending  to  support 
the  doctrine  of  the  over-production  by  the  cell  of  particular  receptive 
atom  groups.  In  this  sense  the  presence  of  gelatin  in  the  culture 
medium  would  be  expected  to  cause  a  more  active  elaboration  of  gelat- 
inase. The  influence  of  gelatin  is  hence  regarded  by  Abbott  and 
Gildersleeve  as  "stimulating  bacteria  to  the  active  production  of 
liquefying  enzymes. " 

My  results  do  not  show  that  gelatinase  production  in  nutrient 
gelatin  always  outstrips  or  exceeds  the  production  in  nutrient  broth. 
The  following  table  illustrates  the  conditions  observed  in  a  series  of 
tests. 

The  table  shows:  (i)  that  some  cultures  develop  gelatinase  more 
quickly  and  abundantly  in  nutrient  broth  than  in  nutrient  gelatin 
prepared  from  the  same  lot  of  broth;  (2)  that  other  organisms  pro- 
duce more  gelatinase  in  nutrient  gelatin  than  in  nutrient  broth;  (3) 
that  in  other  cases  there  is  little  difference.     In  one  instance  (B. 

*  Somewhat  similar  methods  have  been  employed  by  Fermi,  Malfitano  and  others.  Fermi  {Arch.  f. 
Hyg.,  1906,  ss,  p.  140)  has  recently  expressed  a  preference  for  the  use  of  solid  gelatin  in  place  of  fluid 
gelatin  as  a  means  of  testing  the  action  of  these  enzymes,  but  the  disadvantages  of  his  method  seem  to  me 
greater  than  those  of  the  one  here  described.  I  have  not  found  it  difficult  to  obtain  comparable  results 
when  attention  is  paid  to  details  of  mixing,  temperature,  etc.  Some  of  the  experiments  I  have  made- 
could  hardly  be  carried  out  by  the  use  of  solid  gelatin. 

'  Proc.  Roy.  Soc,  1889,  46,  p.  542.  ^  Jour.  Med.  Res.,   1903,   10,  p.  42. 

•  Centralbl.  /.  Bakt.,  1891,   10,  p.  401. 


Experiments  with  Bacterial  Enzymes 


127 


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128  Edwin  O.  Jordan 

subtil  is)  cultures  incubated  at  37 . 5°  gave  more  abundant  production  of 
gelatinase  in  broth  than  in  gelatin,  while  cultures  kept  at  20°  showed 
just  the  reverse.  B.  subtilis  cultures  in  broth  at  37.5°  developed  the 
enzyme  earlier  than  those  at  20°,  while  B.  proteus  cultures  gave  a 
larger  amount  of  enzyme  at  20°  than  at  37  . 5°.  It  is  plainly  incorrect 
to  compare  the  enzyme  content  of  the  liquefied  gelatin  produced  at 
room  temperature  with  that  of  a  whole  flask  of  gelatin  incubated  at 
37°.  On  the  same  ground,  a  comparison  of  broth  and  gelatin  cultures 
at  20°  is  open  to  objection.  Sometimes  old  gelatin  cultures  are  con- 
siderably more  potent  than  the  corresponding  broth  cultures,  but  this 
is  not  invariably  true.  (Table  I,  B.  pyocyaneus,  59  days ;  B.  prodigiosiis, 
39  days.)  This  seems  in  some  cases  to  be  due  to  the  loss  of  the  origi- 
nal strength  of  the  broth  culture  (cf.  B.  amyloruber).  Whether  the 
difference  depends  upon  an  unequal  rate  of  disappearance  of  the 
enzyme  in  the  two  media  or  whether  gelatinase  production  continues 
in  some  cases  in  the  gelatin  culture  after  ceasing  in  the  broth  is  uncer- 
tain. 

The  failure  of  the  presence  of  gelatin  to  provoke  the  formation  of 
gelatinase  does  not  seem  to  be  peculiar  to  bacterial  cultures.  Mal- 
fitano,'  in  working  with  Aspergillus  niger,  found  that  the  kind  of 
enzyme  produced  by  this  mold  did  not  depend  upon  the  presence  of 
gelatin  or  upon  the  nature  of  the  medium,  except  in  so  far  as  this  influ- 
enced the  general  development  of  the  mycehum.  And  Butkewitsch' 
states  that  although  peptone  hinders  the  action  of  the  gelatinase 
formed  by  Aspergillus  and  Penicillium,  this  enzyme  is  produced 
abundantly  in  a  medium  containing  peptone. 

Particular  interest  attaches  to  the  question  of  gelatinase  production 
in  non-proteid  media.,  Fermi^  averred  that  most  bacteria  formed  no 
enzymes  upon  proteid-free  media,  only  B.  pyocyaneus  and  B.  prodi- 
giosus — among  those  tested  by  him — giving  positive  results.  Fermi 
does  not  specify  the  media  employed  further  than  to  state  that  his 
experiments  were  made  "auf  Phosphor- Ammonium-Nahrsalzen  mit 
Zusatz  von  Zuckcr  oder  Glycerin."  The  microbes  mentioned  above 
were  said  to  produce  their  enzymes  only  in  the  media  containing 
glycerin,  not  in  those  with  sugar.  Katz,"*  on  the  other  hand,  was 
unable  to  confirm  Fermi's  observation  that  B.  megatherium  did  not 

■  Ann.  de  I' Inst.  Past.,  1890,   14,  p.  60.  ^  Centralbl.  /.  Bakt.,  1891,  10,  p.  405. 

'  Jahrb,  wiss.  Bot.,  1903,  38,  p.  147.  *  Jahrb.  wiss.  Bot.,   1898,  31,  p.  599- 


Experiments  with  Bacterial  Enzymes  129 

produce  any  enzyme  upon  a  medium  containing  glycerin  without 
peptone.  More  recently  Abbott  and  Gildersleeve'  have  expressed 
the  same  conviction  as  Fermi  regarding  the  production  of  proteolytic 
enzymes  in  non-proteid  media.  They  state  that  in  their  experiments 
the  minimum  evidence  of  digestion  was  given  by  filtrates  from  non- 
protcid  culture  media.  Details  of  these  experiments  are  not  recorded. 
Experiments  made  by  me  show  that  under  proper  conditions  a 
large  amount  of  gelatinase  is  formed  by  some  bacteria  in  non-proteid 
media.     The  following  solutions  were  employed. 

A.  Asparagin o-2g. 

Na,HP04 0.2 

H2O  (redistilled)      .      .      .        loo.oc.c. 

In  this  very  simple  medium  B.  suhtilis  grows  well  and  produces  gelat- 
inase slowly,  but  so  abundantly  that  o .  3  c.c.  of  the  filtrate  of  a  90  day 
culture  at  20°,  o.  i  c.c.  of  a  120  day  culture,  and  0.05  c.c.  of  a  150  day 
culture  brought  about  complete  liquefaction  of  the  standard  amount 
of  gelatin  (p.  125).  The  use  of  the  same  medium  plus  magnesium 
sulphate 

B.  Asparagin o  •  2g. 

Na2HP04 0.1 

MgS04 o.r 

H2O  (redistilled)       .      .      .        loo.oc.c. 

gave  very  similar  results,  but  the  substitution  of  a  potassium  for  the 
sodium  salt 

C.  Asparagin o  ■  2g. 

K2HPO 0.1 

MgS04 0.1 

H2O  (redistilled)        .      .      .        loo.o  c.c. 

led  to  a  negative  result,  as  shown  by  tests  made  with  0.5  c.c.  of  the 
filtrate  of  a  150  day  culture. 


D.     Asparagin 

Na2HP04      .      . 
MgS04     .      .      . 
Dextrose  . 
H,0  (redistilled) 


o .  2  g.  R.     .\sparagin                                  o  •  2  g. 

0.1  NajHP04       ....       0.1 

0 .  I  MgS04 0.1 

1 .  o  Lactose 10 

100. o  c.c.  HjO  (redistilled)  100. o  c.c. 


The  addition  of  dextrose  had  little  effect  in  increasing  the  gelat- 
inase production  in  the  organisms  studied,  B.  suhtilis  in  fact  yielding 
much  less  in  D  than  in  A. 


■  Loc,  cit 


I30 


Edwin  O.  Jordan 

Minimum  Lytic  Dose,  B.  sublilis. 


Age  of  Culture 

A 

D 

60  days           

0.5      C.C.+ 
0.3           + 

0.1           + 
0.05         + 

0.  5  c.c.  0 

go     "     

0.5         0 

03       + 

ICO     "     

03       + 

Lactose,  on  the  other  hand,  is  distinctly  favorable  to  the  formation  of 
the  enzyme. 

Minimum  Lytic  Dose. 


B.  subtilis. . . . 
B.  pyocyaneus .  .( 


Age  of  Culture 


30  days 
60     " 

120     " 
60     " 

120     " 


D 


0.5  c.c. 
o-S 
0.3 
05 

0.5 


O.IO  C.C.+ 

0.05       + 

O.OI  + 

0.5      + 

0.3         + 


The  addition  of  glycerin  (2 .  o  per  cent)  to  C  did  not  give  as  a  rule 
quite  as  strongly  lytic  filtrates  as  lactose,  but  the  difference  was  not 
as  great  as  that  between  lactose  and  dextrose. 

In  the  lactose  medium,  E,  the  M.  L.  D.  was  determined  as  follows: 


B.  sublilis. 


B.  fuchsinus 

B.  pyocyaneus  . . . 
B.  prodigiosus  .  .  . 
B.  ruber  indicus.  . 

B.  proleus 

Sp.  Metchnikovti. 

Sp.  cholerae 


30 

days 

120 

112 

120 

270 

112 

112 

120 

150 

150 

ot 

3 

01 

I 

ot 

3 

I 

5t 


*  This  was  more  strongly  lytic  than  any  broth  or  gelatin  culture  of  this  organism  observed  in  the 
course  of  any  experiment. 

t  This  amount  produced  only  partial  liquefaction. 

These  observations  show  that  in  relatively  simple  synthetic  media 
certain  bacteria  can  produce  large  amounts  of  gelatinase,  amounts  in 
fact  that  are  quite  as  great  as  those  produced  in  broth  or  gelatin  media. 
This  is  true,  however,  as  a  rule  only  when  growth  continues  for  a  long 
period.  The  latter  fact  probably  explains  the  failure  of  observers  to 
discover  the  occurrence  and  extent  of  enzyme  formation  in  non-proteid 
media. 

Reaction  0}  medium. — The  influence  of  the  reaction  of  the  cul- 
ture medium  upon  enzyme  production  is  advantageously  studied  in 
the  case  of  such  an  organism  as  B.  pyocyaneus.    As  is  well  known, 


Experiments  with  Bacterial  Enzymes  131 

this  bacillus  is  an  active  alkali-former.  The  reaction  of  neutral  broth 
inoculated  with  B.  pyocyaneus  becomes  speedily  alkaline,  while  gela- 
tin inoculated  at  the  same  time  becomes  acid,  owing  to  the  acidigenic 
action  of  the  gelatinolytic  enzyme.  The  two  cultures  accordingly 
diverge  in  their  reaction.  Enzyme  formation,  however,  occurs  in 
both  media  and  apparently  takes  place  to  about  the  same  extent 
in  each.  Parallel  cultures  of  B.  pyocyaneus  in  broth  and  gelatin 
reacted  as  follows  after  24  days  at  36°:  gelatin  4.0  per  cent  acid; 
broth  0.5  per  cent  alkaline.  One-tenth  c.c.  of  fihrate  from  each 
culture  gave  complete  liquefaction;  o.oi  c.c.  was  negative  for  the 
broth  culture,  but  the  same  amount  of  the  gelatin  filtrate  produced 
a  slight  softening.  In  another  gelatin  culture,  26  days  old  at  20°, 
alkali  formation  had  almost  completely  neutralized  the  acids  arising 
from  the  gelatin  splitting,  and  the  filtrate  in  this  case  reacted  neutral 
to  phenolphthalein.  The  gelatinolytic  potency  of  this  filtrate,  how- 
ever, was  almost  exactly  the  same  as  in  the  instance  just  cited,  viz. : 

*o.  I  c.c + 

0.05 d= 

003 - 

0.01 o 

*In  aU  the  tables  the  following  signs  are  used:   +=  complete  liquefaction;    ±  =  partial  liquefaction 
—  =  gelatin  noticeably  softened;  o  =  perfectly  solid,  like  control. 

In  another  case  a  culture  of  B.  pyocyaneus  in  broth  was  compared 
with  a  control  culture  in  gelatin  with  the  following  result : 

Forty-eight  Hours  at  37°. 


Broth  (-f-0.2%) 

Gelatin  (+0.5%) 

0.5  c.c 

-1- 
-1- 
0 

+ 

0,3          

+ 

0,1              

Nine  Days  at  37°. 


0.05    c.c. 

0.03 

0.01 

0.005 

0.003 


Gelatin  (+2.5%) 


It  is  evident  from  these  and  manv  other  observations  of  the  writer 
in  respect  to  varying  reactions  among  gelatinolytic  filtrates  that  the 
production  of  a  gelatin-liquefying  enzyme  is  not  very  dependent  upon 


132  Edwin  O.  Jordan 

the  reaction  of  the  culture  medium.  It  is  probable  that  the  reaction 
is  of  importance  only  in  so  far  as  it  influences  the  general  conditions 
of  growth  of  the  microorganism.  The  facts  to  be  adduced  presently 
concerning  the  conditions  under  which  the  enzymes  manifest  their 
activity  tend  to  support  this  view. 

PHYSICAL    CHARACTERS. 

Resistance  of  gelatinases  to  heat. — The  statements  of  authors  are 
not  in  harmony  on  this  point.  Fermi'  found  that  the  gelatinolytic 
enzymes  were  destroyed  at  relatively  low  temperatures :  the  majority 
of  those  tested  by  him  were  rendered  inactive  at  5o°-55°  C,  and  all 
were  destroyed  at  7o°C.  Abbott  and  Gildersleeve,^  on  the  other 
hand,  observed  a  surprising  heat  resistance  on  the  part  of  the  proteo- 
lyitc  enzymes  elaborated  by  certain  species,  and  asserted  that  some 
were  even  "capable  of  exhibiting  their  characteristic  function  after 
exposure  in  the  moist  state  to  a  temperature  of  100°  C.  for  15  to  30 
minutes."  Hata^  also  declares  that  the  enzymes  of  B.  prodigiosus 
and  B.  fluorescens  liq.  resist  high  temperatures.  None  of  these 
authors  have  given  definite  data  concerning  the  amount  and  strength 
of  the  enzymes  employed  in  their  experiments. 

The  varying  results  obtained  in  some  of  my  earlier  work  gave  rise 
to  the  suspicion  that  differences  in  the  reaction  of  the  medium  in 
which  the  enzyme  was  contained  were  responsible,  at  least  in  part,  for 
the  divergent  statements.  It  has  been  pointed  out  elsewhere  in  this 
paper  that  the  reaction  of  the  culture  medium  is  often  profoundly 
affected  by  bacterial  growth,  that  gelatin  cultures  of  liquefying  species 
are  more  acid  than  the  corresponding  broth  cultures,  and  that  the 
reaction  varies  according  to  the  particular  stage  of  growth  and  enzyme 
action  at  which  it  is  tested.  The  influence  of  the  reaction  of  the 
medium  (presence  of  H  or  OH  ions)  upon  the  thermal  death-point  of 
the  enzyme  is  shown  in  the  experiments  which  follow.  It  is  unneces- 
sary further  to  multiply  such  instances.  In  almost  every  case  tested 
it  was  found  that  increasing  the  acidity  of  the  enzyme-containing 
fluid  raised  the  "  heat  resistance"  of  the  enzyme,  while  adding  to 
the  alkalinity  lowered  it.  In  one  case  the  M.  L.  D.  of  an  enzyme 
(o.oi  c.c,  B.  prodigiosus)  was  not  affected  by  boiling  for  15  minutes 

'  Centralbl.  f.  Bakt.,  1891,  10,  p.  401.  '  Cenlralbl.  f.  Bakt.,   1904,   Ref.  34,  p.   308. 

•  Jour.  Med.  Res.,  1903,  s,  p.  42. 


Experiments  with  Bacterial  Enzymes 


133 


in  a  5  per  cent  acid  medium,  while  in  a  neutral  solution  50  M.  L. 
D.  were  completely  destroyed.  That  acid  and  alkali  are  not  the 
only  substances  whose  presence  must  affect  the  "heat  resistance"  of 


EXPERIMENT. 
Broth  Culture  B.  prodigiosus. 

UNHEATED. 


Amt.  of  Culture  c.  c. 

2.5%   Acid 

1%  Acid 
Grig.  React. 

Neutral 

2.5%  Alka- 
line 

0.2 

+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 
± 

+ 
+ 
4- 
+ 
0 

+ 

0.1 

+ 

0.06 

0.03 

0. 01 

+ 
+ 
0 

HEATED    7a 

"  FOR   I   HOUR 

* 

0.2 

+ 
+ 
+ 
± 
0 

+ 
± 
0 
0 
0 

0 
0 
0 
0 
0 

0 

0.1 

0 

0 .  06 

0 

0.0^ 

0 

o.oi 

0 

*In  each  case  2  c.c.  of  culture  or  filtrate  was  placed  in  a  sealed  tube  and  immersed  in  a  water-bath. 

EXPERIMENT. 
Broth  Culture  B.  pyocyaneus. 

UNHEATED. 


Amt.  of  Culture  c.c. 

3%  Acid 

1.3%  Acid 
Original 

Neutral 

0.  20 

0.08 

0.06 

0.04 

-t- 
-1- 
-f- 
0 

+ 
+ 
-1- 
+ 
0 

+ 
+ 
+ 
+ 
0 

HEATED    TO    70°    FOR    30   MINUTES, 


0.20. 
0.08. 
0.06. 
0.04. 
0.02. 


an  enzyme  is  obvious.  In  some  cases  a  neutral  broth  culture  was 
found  to  resist  heating  better  than  a  neutral  gelatin  culture,  that  is, 
2  M.  L.  D.  of  the  gelatin  culture  were  destroyed  at  a  lower  tempera- 
ture. Among  the  great  variety  of  substances  produced  by  bacteria, 
there  must  be  many  whose  presence  affects  the  stability  of  the  enzyme 
when  heat  is  applied.  Any  accurate  determination  of  the  heat  resist- 
ance of  bacterial  enzymes  is  hardly  possible  when  the  enzymes  are 


134 


Edwin  O.  Jordan 


heated  in  the  cultures  in  which  they  are  produced,  and  even  when 
the  enzymes  are  separated  out  as  far  as  possible,  there  remains  a  pos- 
sible source  of  error  in  the  adherent  impurities. 

Filtration  of  gelatinase. — Levy'  has  shown  that  certain  enzymes, 
rennet,  for  example,  are  retained  by  the  Berkefeld  and  Chamberland 
bougies,  while  others  pass  through  these  filters.  A  few  experiments 
have  been  made  to  determine  how  far  gelatinase  is  removed  by  filtra- 
tion. A  31  day  culture  of  B.  pyocyaneus,  grown  at  37°  and  reacting 
0.9  per  cent  alk.  was  filtered  at  2o°C.  with  the  following  result: 


Berkefeld  Bougie 
60  m.  X15  m. 
One  filtration 


0.05  c.c 

0.03 

0.02 


Another  trial  with  a  different  culture  of  B. 
follows : 


pyocyaneus  was  as 


0.0s  c.c 

0.03 

0.02 

O.OI 


Unfiltered 


+ 
+ 
+ 


Berkefeld  Bougie 

6om.Xsom. 

One  filtration 


+ 
+ 
+ 


Chamberland  Bougie 
200  m.  X20  m. 
One  filtration 


+ 
-1- 
-1- 


Several  successive  passages  through  a  Berkefeld  bougie  did  not 


remove  the  gelatinase. 


Unfiltered 

Berkefeld  Bougie. 

69  m.  X15  m. 

2  Filtrations 

4  Filtrations 

0.02  c.c 

O.OI            ...           .     . 

+ 
± 

+ 
± 

+ 

A  similar  result  was  obtained  with  B.  subtilis.     (Broth  culture, 
8  days.) 


Unfiltered 

Chamberland  Bougie 
200  m.  X20  m. 
One  filtration 

0. 1  c.c 

+ 
± 

0 

+ 

± 

0.3        

O.I            

0 

Jour.  Infect.  Dis.,  1905,  2,  p.  i. 


Experiments  with  Bacterial  Enzymes  135 

In  respect  to  passage  through  the  Berkefcld  bougie,  therefore,  the 
bacterial  gclatinases  agree  with  ptyaUn  and  taka-diastase  rather  than 
with  rennet  (cf.  Levy,  op.  cit.). 

CONDITIONS    OF    ENZYME    ACTIVITY. 

Reaction — It  is  well  known  that  the  acid  or  alkahne  reaction  of 
the  medium  in  which  an  enzyme  is  present  often  exercises  great 
influence  upon  the  activity  of  the  enzyme.  As  regards  the  parti- 
cular enzyme  under  consideration,  it  seems  to  have  been  generally 
assumed  that  the  gelatin-liquefying  enzymes  produced  by  bacteria 
were  most  potent  in  an  alkahne  medium.  Thus  Abbott  and  Gilder- 
sleeve'  state: 

As  is  the  case  for  the  majority  of  proteolytic  enzymes,  be  their  origin  wha 
it  may,  we  find  our  filtrates  to  be  uniformly  more  active  when  they  are  of  alkaline 
than  of  neutral  or  acid  reaction.  When  acidified  they  are  as  a  rule  inactive.  In  a 
simlar  manner  their  production  by  the  growing  organism  is  always  more  marked  in 
alkaline  than  in  either  neutral  or  acid  media,  even  though  the  latter  is  not  sufficient 
to  depress  growth  to  any  marked  extent. 

Further  details  are  not  given  in  their  paper. 

The  statement  that  gelatinase  production  is  more  marked  in  alka- 
line than  in  neutral  or  acid  media,  evidently  needs  some  qualification 
in  view  of  the  facts  set  forth  in  another  part  of  this  paper  (p.  130). 
It  by  no  means  follows  that  the  initial  reaction  of  the  culture  medium 
represents  the  conditions  under  which  the  enzyme  is  produced.  i\ 
gelatin  culture  and  a  broth  culture  of  a  liquefying  species  diverge  in 
respect  to  reaction  from  the  moment  growth  begins  to  take  place,  the 
gelatin  culture  invariably  becoming  more  acid. 

The  fact  that  gelatin  liquefied  by  enzyme  action  has  a  strongly  acid 
reaction  seems  to  have  been  generally  overlooked  by  bacteriologists.* 
The  products  of  gelatin  digestion  comprise  glycocoU,  aspartic  acid, 
and  glutaminic  acids,  and  other  acid  substances.  When,  therefore, 
any  one  of  the  bacterial  gelatinases,  or,  for  that  matter,  Griibler's 
pancreatin,  is  added  to  gelatin,  the  substances  produced  by  the  enzyme 
action  impart  a  strongly  acid  reaction  to  the  liquefied  mass.  In  most 
liquefied  gelatin  cultures  of  bacteria  the  reaction  ranges  as  high  as 
2.0  per  cent  to  3 .0  per  cent  acid  to  phenolphthalcin,  and  in  some  cases 
it  is  over  4.0  per  cent.     By  the  v^ery  conditions  of  its  action,  then,  a 

'  Loc.  cit.,   p.  47. 
♦  I  have  discussed  this  elsewhere,  Science,  February  q,  1906,  p.  j2o. 


136 


Edwin  O.  Jordan 


bacterial  gelatinase  must  exercise  its  effect  almost  exclusively  in  an 
acid  medium. 

The  change  in  the  reaction  of  the  medium,  as  might  be  expected, 
is  not  confined  to  the  hquefied  gelatin,  but  is  communicated  by  diffu- 
sion to  the  yet  unliquefied  portions. 

EXPERIMENT. 

Two  hundred  c.c.  of  10  per  cent  nutrient  gelatin  (neutral)  were  placed  in  600  c.c" 

flasks.     These  were  inoculated  on  one  side  and  the  flasks  tipped  so  that  liquefaction 

took  place  in  only  one-half.     After  incubation  at  20°  C.  for  four  days   the   liquefied 

gelatin  was  drawn  off  and  the  reaction  of  this  and  of  the  unliquefied  portion  determined. 

B.  amyloruher. 

Liquefied  gelatin i .  8%  acid 

Solid  gelatin 0.7        " 

B.  suUilis. 

Liquefied  gelatin 2 . 9%  acid 

Gelatin  removed  from  directly  under  liquefied  area  .      .      .0.9         " 

Gelatin  taken  3  cm.  from  liquefied  region 0.6        " 

Gelatin  from  opposite  side  of  flask 0.4        " 

Perhaps  one  reason  why  the  standardization  of  nutrient  gelatin  for 
plate  cultures  has  not  been  so  successful  as  could  be  desired  is  because 
after  growth  begins  alterations  in  reaction  occur  in  varying  degrees 
according  to  the  relative  abundance  or  scarcity  of  hquefying  species. 

It  follows,  too,  that  the  reactions  produced  by  bacteria  in  ordinary 
broth  are  dependent,  not  only  upon  the  presence  of  sugar,  but  to  a 
degree  upon  the  amount  of  gelatin  and  similar  substances  in  the 
medium. 

EXPERIMENT. 
Neutral  gelatin  was  inoculated  with  B.  pyocyaveus  and  the  filtrate  was  found 
after  four  days  at  37°  C.  to  be  i  .8  per  cent  acid.      The  action  of  the  filtrate  (18  hrs.) 
upon  carbol  gelatin  of  different  degrees  of  alkalinity  was  as  follows: 


Amount  of  Filtrate 


1.8%  Acid- 
0.5  c.c. 
o 
o 
o 
o 
o 

2% 

o 
o 
o 
o 
o 
o 


3  

I  

05       

03       

Alk.  witii  N/V  NaOH— ' 
5    c.c 

3  

I  

05         

03         

01 


Carbol  Gelatin 


0.7%  Acid 


+ 
+ 
+ 
+ 
± 
o 


Neutral 


+ 
± 


± 
± 
± 

o 
o 


0.7%  Alk. 


+ 


o 
o 

± 
± 

o 
o 
o 

o 


Experiments  with  Bacterial  Enzymes 


137 


The  particular  question  as  to  the  effect  of  the  initial  reaction  of 
the  medium  upon  enzyme  production  was  thus  answered  in  a  some- 
what unexpected  manner. 

This  experiment  was  repeated  with  another  lot  of  carbol  gelatin, 
0.6  per  cent  acid,  neutral,  and  0.5  per  cent  alkahne  respectively, 
with  the  same  result,  namely,  the  acid  and  neutral  gelatins  were  more 
quickly  affected  by  the  enzyme  than  the  alkaline  gelatin.  A  com- 
parison of  B.  pyocyaneus  gelatinase  and  Griibler's  pancreatin  (5  per 
cent  solution  in  distilled  water)  resulted  as  follows: 


EXPERIMENT. 


Carbol  Gelatin 

0 . 8%  Acid 

Neutral 

0.8%  Alkaline 

B.  pyocyaneus — 
0.3    c.c 

+ 
+ 

0 

+ 
+ 
=*= 
0 
0 

+ 

+ 

0 

+ 
+ 
± 

0 

^ 

0.1           

0.0s         

0.03         

Pancreatin  solution — 
0.03  c.c 

+ 

OCX          

+ 

0.005       

^ 

0 . 003       

0.001        

The  gelatinases  produced  by  B.  amyloruher  and  Sp.  Finkler-Prior 
behaved  as  follows: 


experiment. 


Amount  of  Filtrate 


B.  amyloTuber- 
0.3  c.c.  .  .  . 
0.1 


Sp. 


OS  

03  

01  

Finkler-Prior- 

30  c.c 

10         

05         

03         

01  


Carbol  Gelatin 


i%~Acid 


+ 
± 
± 


Neutral 


+ 


+ 
+ 


1%  Alkaline 


+ 
± 


+ 
+ 


Further  experiments  with  gelatin  of  a  wider  range  of  acidity  and 
alkahnity  gave  the  following  results: 


138 


Edwin  O.  Jordan 

EXPERIMENT. 


Amount  of 

Carbol  Gelatin 

Filtrate 

5%  Acid 

3%  Acid 

2%  Acid 

Neutral 

2%  Alk. 

3%  Alk. 

B.  pyocyaneus — 
I  .0  c.c 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 

0 
0 

0 
0 
0 

0 
0 
0 
0 
0 

+ 

0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 

+ 
+ 
+ 

0 
0 
0 

+ 
4- 
+ 
+ 
+ 
0 

+ 

+ 

'  + 

0 

+ 
+ 
+ 
+ 
+ 
± 
0 

+ 

0 
0 
0 

0 

+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 

+ 

0 

+ 

OS         

04         

03         

0.2         

0.1          

Sp.  Finkler-Prior — 
0.  -i  c.c 

0 
0 

0 
0 
0 

+ 

0.2          

0.1          

0 .  08       

o.os        

B.  prodigiosu.i — 
0.  %  c.c 

+ 
+ 
+ 
+ 

0.1         

0.06       

0 .  04       

0.02        

0.01        

0.005     

4- 
+ 
+ 
± 
0 
0 

These  experiments  show  that  not  all  gelatin-liquefying  enzymes 
area  like  in  respect  to  the  conditions  of  their  activity ;  that  some  liquefy 
a  neutral  gelatin  quite  as  rapidly  as,  or  even  better  than,  a  slightly 
alkaline  one  (B.  amyloruber,  B.  prodigiosus);  that  some  liquefy  even 
more  rapidly  in  an  acid  medium  than  in  an  alkaline  one  (B.  pyocya- 
neus); and  that  in  one  case  alkaline  carbol  gelatin  is  Hquefied  more 
rapidly  than  a  neutral  gelatin  {Sp.  Finkler-Prior).  Vines'  has 
recently  shown  that  the  digestion  of  fibrin  by  vegetable  proteases 
occurs  in  some  cases  with  both  acid  and  alkaline  reaction  and  in 
others  is  limited  to  acid  reaction.  He  interprets  his  results  as  indi- 
cating the  existence  of  two  distinct  vegetable  proteases,  one  of  which 
belongs  to  the  peptases,  although  the  "vegetable  pepsin"  differs 
from  animal  pepsin  in  being  able  in  some  cases  to  act  in  an  alkaline 
medium. 

I  have  already  pointed  out  that  the  acid  reaction  developed  in  the 
course  of  gelatin  digestion  interferes  with  any  strict  evaluation  of 
the  influence  of  reaction  upon  the  work  of  the  gelatinases.  For 
example,  tubes  of  carbol  gelatin  with  an  initial  reaction  2  per  cent 
alkaline  were  liquefied  in  20  hours  by  the  enzymes  of  B.  prodigiosus 
and  Sp.  Finkler-Prior  respectively,  and  both  tubes  then  had  a  reaction 
of  but  0.4  per  cent  alkaline.     By  the  action  of  trypsin  for  20  hours 

'  Annals  of  Botany,   1905,   19,  pp.  149-87. 


Experiments  with  Bacterial  Enzymes  139 

at  37°  C.  carbol  gelatin  originally  0.05  per  cent  alkaline  was  made 
acid  as  follows: 

S%  Trypsin  Sol.  Carbol  Gelatin 

0.003  c.c 0.1%  acid 

0.03  i.o         " 

01  2.3 

All  the  bacterial  gclatinascs  tested  are  able  to  manifest  their  activity 
in  a  medium  more  or  less  acid.  It  is  further  evident  from  the  experi- 
ments cited  above  that  the  initial  reaction  of  the  gelatin  is  not  a  matter 
of  indifference,  that  the  gelatinases  derived  from  several  bacterial 
species  are  not  alike  in  this  respect,  and  that  it  is  certainly  not  true 
that  an  initial  alkaline  reaction  presents  in  all  cases  the  most  favorable 
conditions  for  the  gelatinolytic  process. 

Temperature. — The  temperatures  at  which  the  enzymes  manifest 
their  maximum  activity  have  been  approximately  determined  in  several 
cases.  In  every  instance  the  enzymes  have  produced  greater  lique- 
faction at  37.5°  than  at  lower  temperatures.  This  is  true  even 
when  the  microorganism  forming  the  enzyme  grows  better  at  a  lower 
temperature.  A  strain  of  Sp.  Finkler- Prior,  for  example,  that  grew 
well  at  20°  C,  but  refused  to  grow  at  37°  C,  gave  rise  to  an  enzyme  that 
liquefied  more  rapidly  at  37°  than  at  20°,  more  rapidly  at  45°  than  at 
37°,  more  rapidly  at  56°  than  at  45°,  and  even  at  60°  liquefied  better 
than  at  37°,  though  not  so  well  as  at  56°. 

The  following  results  were  obtained  with  a  B.  pyocyaneus  culture 
(M.  L.  D.  at  37°  C,  20  hours  was  o.oi  c.c): 


Amount  of  Filtrate 


0.1    c.c. 

0.08 

COS 

0.03 

0.01 


One  HonR  At 

30° 

37' 

45° 

55' 

60' 

+ 

+ 

4- 

+ 

+ 

— 

— 

± 

+ 

+ 

0 

— 

— 

± 

— 

0 

0 

— 

— 

0 

0 

0 

■^ 

■— ~ 

0 

-O 


Another  test  with  a  pyocyaneus  gelatinase  showed  that,  as  in  the 
experiment  just  cited,  slightly  more  liquefaction  was  produced  at  45 
than  at  37°;  at  65°,  however,  the  activity  of  the  enzyme  was  distinctly 
checked  but  not  altogether  inhibited. 

The  gelatinase  of  B.  prodigiosus  behaved  in  a  similar  fasliion  (M. 
L.  D.  20  hours  at  37°  was  o .  02  c.c.) : 


140 


Edwin  O.  Jordan 


Amount  of  Filtrate 

One  Hour  at 

37° 

45° 

56" 

O.  5  c.c 

+ 
+ 
+ 

o 
o 

+ 
+ 
+ 
± 

o 

+ 

O  -I         



o.i         

o 
o 

o 

These  experiments  show  that  at  least  in  some  cases  the  bacterial 
gelatinases  exhibit  their  maximum  effect  at '  temperatures  consider- 
ably above  the  optimum  temperatures  for  the  growth  of  the  organism 
that  produces  them.  The  enzyme  activity  may  even  be  manifested 
above  the  thermal  death-point  of  the  bacteria  producing  the  enzyme 
(cf .  pyocyaneus  gelatinase  at  6o° — the  thermal  death-point  of  B.  pyO' 
cyaneus  is  56° — and  Sp.  Finkler- Prior). 

THE    ACTION    OF   FORMALIN   UPON   LIQUEFIED   GELATIN. 

Mavrojannis'  has  called  attention  to  what  he  considers  an  impor- 
tant fact,  namely,  that  while  formahn  has  a  solidifying  action  upon 
the  gelatin  liquefied  by  certain  bacteria,  the  gelatin  liquefied  by  other 
bacteria  remains  permanently  fluid,  even  when  subjected  to  the  influ- 
ence of  formalin  for  long  periods.  Mavrojannis  explains  the  dififer- 
ence  by  supposing  that  different  stages  occur  in  the  destruction  of  the 
gelatin  molecule,  and  that  the  kind  of  enzyme  that  is  produced  by 
certain  microorganisms  stops  short  with  the  production  of  gelatoses 
(hardened  by  formalin),  while  in  other  cases  a  different  enzyme  is 
produced  that  continues  the  digestion  to  the  formation  of  gelatin- 
peptones  (not  hardened  by  formalin).  In  the  former  category  are  to 
be  put,  according  to  Mavrojannis,  St.  pyog.  aureus,  St.  pyog.  alhus, 
B.  anthracis,  B.  pyocyaneus,  and  Sp.  cholerae,  while  in  the  latter 
belong  Sp.  Denecke,  Sp.  Finkler-Prior,  and  Sp.  Metchnikovii. 

I  have  used  the  same  methods  for  hardening  as  those  employed  by 
Mavrojannis,^  namely,  introduction  of  the  liquefied  cultures  (gelatin, 
10  per  cent)  into  a  tightly  closed  jar  in  the  bottom  of  which  is  a  40  per 
cent  formahn  solution;  the  gas  hberated  from  this  solution  brings 
about  the  hardening  in  a  constant  and  uniform  fashion.  I  have  also 
added  to  2  c.c.  of  the  liquefied  gelatin  five  drops  of  formalin  and  then 
placed  the  tubes  in  the  formalin  jar.     The  outcome  is  about  the  same 


■  Ztschr.  I.  Hyg.,  1903,  45,  p.  108 


'  Op.  cit.,  p.  109. 


Experiments  with  Bacterial  Enzymes  141 

in  the  two  procedures,  except  that  the  addition  of  formahn  accelerates 
the  hardening  process.     The  results  may  be  stated  briefly : 

Eight  different  species  have  been  tested:  B.  pyocyaneus  (nine 
strains),  B.  anthracis,  B.  prodigiosus,  B.  subtilis,  Sp.  cholerae,  Sp. 
Finkler- Prior,  Sp.  Metchnikovii,  and  Sp.  Denecke.  In  each  case  the 
resuhs  were  the  same.  Young  gelatin  cultures  became  solid  in  the 
formahn  jar,  while  older  cultures  of  the  same  species  remained  fluid 
even  after  exposure  to  the  formalin  vapor  for  three  months.  Cultures 
grown  at  20°  always  solidified  sooner  than  those  of  the  corresponding 
age  grown  at  37°.  Different  strains  of  the  same  microorganism  gave 
different  results.  For  example,  the  gelatin  liquefied  by  nine  strains 
of  B.  pyocyaneus,  all  grown  at  20°  for  21  days,  hardened  in  formalin 
in  24  hours  in  one  case  and  in  48  hours  in  two  cases,  but  was  still  fluid 
after  three  months  in  the  other  six.  The  gelatin  liquefied  by  a  three- 
day  growth  of  B.  prodigiosus  at  20°  solidified  in  three  days,  but  the 
15  day  growth  of  the  same  organism  remained  fluid  at  the  end  of 
three  months. 

Mavrojannis'  has  even  gone  so  far  as  to  champion  the  action  of 
formalin  upon  Hquefied  gelatin  cultures  as  a  means  of  distinguishing 
the  cholera  vibrio  from  other  species.  According  to  this  writer,  Sp. 
cholerae  manufactures  a  gelatinase  which  is  capable  of  digesting  gela- 
tin only  as  far  as  the  stage  of  gelatoses  (solidifying  in  formalin),  while 
Sp.  Metchnikovii,  Sp.  Denecke,  and  Sp.  Finkler-Prior  push  the  de- 
composition to  the  gelatin-peptone  stage  (permanently  liquid).  An 
experiment  with  the  cultures  of  these  organisms  in  the  laboratory 
collection  gave  the  following  results : 

EXPERIMENT. 

A. 

Cultures  grown  at  20°  for  10  days,  then  transferred  to  formalin  jar;  liquefaction 

approximately  the  same  in  all. 

Sp.  Denecke Hard  in  24  hours 

Sp.  Metchnikovii "     "  48    " 

Sp.  cholerae  (Wherry,  Manila) "     "   14  days 

Sp.  Finkler-Prior Not  hard  in  106     " 

B. 

Cultures  grown  at  37.  5°  for  6  days,  then  transferred  to  formalin  jar. 

Sp.  Denecke  1 

sp.  Metchnikovii  ^,i  ,j     jj  ^j^^.^  no  days 

Sp.  cholerae  (Wherry,  Manila)  ^ 

Sp.  Finkler-Prior  ' 

'  Jour,  de  physiol.  el  Path,  ginir.    1904,  6,  p.  J73 


142  Edwin  O.  Jordan 

This   experiment   gives   no   support   to    Mavrojannis'    criterion    of 
differentiation. 

The  only  conclusion  that  can  be  drawn  from  all  my  observations 
on  this  matter  is  that  no  fundamental  distinction  between  the  different 
bacterial  gelatinases  exists  in  the  sense  alleged  by  Mavrojannis. 
Young  gelatin  cultures  and  those  liquefied  by  feeble  strains  solidify 
when  subjected  to  the  action  of  formalin;  the  same  is  true  of  some 
cultures  grown  at  20°  as  compared  with  those  of  the  same  species 
grown  at  37°.  On  the  other  hand,  all  the  old  cultures  of  vigorously 
liquefying  strains,  of  whatever  species,  are  not  hardened  by  formalin 
action.  In  other  words,  the  difference  observed  is  simply  one  of  degree 
and  not  of  kind.^ 

RELATION   BETWEEN   BACTERIAL   GELATINASES   AND   BACTERIAL 

HEMOLYSINS. 

The  question  has  been  raised  recently  as  to  whether  the  hemolytic 
action  displayed  by  certain  bacterial  filtrates  is  not  simply  one  mani- 
festation of  their  proteolytic  activity.  On  this  assumption  the  libera- 
tion of  the  hemoglobin  is  regarded  as  due  to  the  action  of  an  enzyme 
upon  the  stroma  of  the  erythrocytes.  Abbott  and  Gildersleeve,^  as 
the  result  of  their  studies,  reached  the  conclusion  that  "one  may  as 
reasonably  attribute  the  hemolysis  exerted  by  these  filtrates  to  the 
action  of  their  proteolytic  enzymes  upon  the  stroma  of  the  erythro- 
cytes as  to  any  other  factor."  These  authors  take  the  liquefaction  of 
gelatin  as  the  criterion  of  proteolytic  action,  and  base  their  belief  on 
the  identity  of  the  hemolytic  and  gelatin-liquefying  properties  upon 
certain  general  analogies.  Opposed  to  this  view  are  the  observations  of 
Buxton^  and  Eijckman,4  who  found  that  the  destruction  of  blood 
corpuscles  in  blood-agar  does  not  proceed  pari  passu  with  the  lique- 
faction of  gelatin.  The  fact  that  many  bacteria,  such  as  B.  coli,. 
B.  typhosus,  and  others,  that  are  unable  to  liquefy  gelatin  can  exert 
a  hemolytic  action,  has  been  considered  an  additional  reason  for  not 
identifying  the  gelatin-liquefying  and  hemolytic  power  of  bacterial 
filtrates. 

'  Since  writing  the  above,  a  paper  by  Tiraboschi  {Ann.  d'  igiene  sperim.,  151,  905,  p.  429;  Abstr., 
Bull,  de  rinsl.  Pasl.,  1905,  3,  p.  922)  has  appeared  which  supports  this  position. 

'  Jour.  Med.  Res.,  1903,  10,  42.  *  Centralbl.  /.  Bakt.,   1901,  20,  p.  841. 

3  Amer.  Med.,  July  25,  1903,  p.  137. 


Experiments  with  Bacterial  Enzymes 


M3 


The  following  facts  constitute  further  and  apparently  incontrover- 
tible evidence  that  the  hemolytic  and  gelatinolytic  substances  are,  at 
least  in  a  number  of  bacterial  filtrates,  entirely  distinct.  While  it  is 
true  that  the  potency  of  gelatinase  in  some  cases  is  not  entirely 
destroyed  by  heating  to  ioo°  C.  for  lo  to  15  minutes  (B.  pyocyaneus 
and  B.  prodigiosus),  it  is  also  true  that  heating  for  30  minutes  at  110°  C. 
destroys  completely  all  the  power  of  these  filtrates  to  liquefy  gelatin, 
but  leaves  absolutely  intact  their  hemolytic  power.  In  the  second 
place,  certain  filtrates  which  possess  marked  ability  to  liquefy  gelatin 
may  be  entirely  devoid  of  hemolytic  power.  A  single  instance  may 
be  given.  A  seven-months-old  culture  of  B.  prodigiosus  in  asparagin- 
phosphate-sulphate-sucrose  solution  yielded  a  filtrate,  0.05  c.c.  of 
which  liquefied  a  tube  of  gelatin  completely  in  16  hours  at  37.5°. 
Such  a  solution  has  some  osmotic  action  on  dog  corpuscles,  but  the 
addition  of  o .  4  per  cent  NaCl  renders  it  isotonic,  although  not  affect- 
ing its  power  to  liquefy  gelatin.  The  filtrate  is  then  strongly  gelat- 
inolytic, but  has  no  hemolytic  effect  on  dog  or  rabbit  corpuscles, 
even  when  0.5  c.c.  is  used. 

Other  examples  may  be  presented  in  tabular  form : 


Filtrate 


Gelatinolvsis 


Hemolysis  (Dog 
Corpuscles) 


From  3  months'  old 
broth  culture — 


f  Sp.  Finkler-Prior 

B.    amyloruber 

From  7  mos.'  old  broth  culture — B.  Pyocyaneus . 


0.8  c.c. 
o.  I 
0.05 
°-5 

o.  5 


None 

Complete 
None 


Complete 

None 

\'ery  strong 


It  is  true,  then,  (i)  that  certain  hemolytic  filtrates,  heated  at  110° 
may  be  robbed  completely  of  their  power  to  liquefy  gelatin  without 
evincing  any  diminution  of  hemolytic  power  {B.  pyocyaneus,  B. 
prodigiosus);  (2)  that  a  bacterial  filtrate  may  possess  gelatinolytic 
power  without  being  able  to  produce  any  hemolysis  whatsoever  {B. 
amyloruber);  (3)  that  a  bacterial  filtrate  may  be  strongly  hemolytic 
without  possessing  any  power  to  liquefy  gelatin. 

SUMMARY   AND   CONCLUSIONS. 

I.  There  is  no  evidence  that  the  presence  of  gelatin  in  a  culture 
medium  leads  to  any  particularly  rapid  or  abundant  production  of 
the  specific  ferment  acting  upon  the  gelatin.     On  the  contrar)-  other 


144  Edwin  O.  Jordan 

factors  are  of  much  greater  influence  than  the  presence  of  gelatin  in 
determining  the  generation  of  liquefying  enzymes. 

2.  In  simple  non-proteid  solutions  of  asparagin,  lactose,  and 
mineral  salts  (sodium  phosphate  and  magnesium  sulphate)  gelatinase 
is  produced  by  some  bacterial  species  quite  as  abundantly,  although 
generally  not  as  rapidly,  as  in  nutrient  broth  or  gelatin.  Lactose  is 
more  favorable  than  dextrose  to  gelatinase  production. 

3.  The  reaction  of  the  culture  medium  is,  at  least  in  some  cases, 
without  apparent  effect  upon  the  enzyme  production  except  as  it 
afifects  the  conditions  of  bacterial  growth. 

4.  The  heat  resistance  of  the  gelatinase,  as  this  is  determined  by 
heating  the  ordinary  fluid  culture,  is  conditioned  by  a  variety  of  influ- 
ences. One  of  these  is  the  reaction  of  the  medium.  The  gelatin- 
liquefying  enzymes  produced  by  a  number  of  microorganisms  endure 
heat  very  much  better  when  heated  in  an  acid  than  in  an  alkaline  or 
a  neutral  medium.  The  usual  tests  of  heat  resistance  of  bacterial 
enzymes  which  are  made  directly  with  the  culture  in  which  the 
enzymes  are  produced  have  little  value. 

5.  Some,  at  least,  of  the  bacterial  gelatinases  pass  through  the 
Berkefeld  filter  without  weakening. 

6.  The  reaction  most  favorable  to  the  manifestation  of  gelatino- 
lytic  activity  is  different  in  different  cases.  The  enzymes  produced 
by  some  species  act  most  rapidly  in  a  medium  slightly  acid  to  phenol- 
phthalein,  while  others  do  best  with  an  alkaline  reaction.  It  can  no 
longer  be  maintained  that  an  initial  alkaline  reaction  affords  the  opti- 
mum condition  for  all  bacterial  proteolytic  enzymes. 

7.  The  enzymes  that  have  been  experimented  with  act  more  ener- 
getically at  45°  C.  than  at  lower  temperatures.  They  may  continue 
to  be  effective  at  temperatures  as  high  as  60°. 

8.  Some  bacterial  enzymes  manifest  their  activity  at  temperatures 
considerably  above  the  thermal  death-point  of  the  organism  produ- 
cing them. 

9.  The  gelatin  liquefied  by  some  cultures  of  bacteria  is  hardened 
by  formalin.  This,  however,  is  true  chiefly  in  the  case  of  young  cul- 
tures, of  cultures  grown  at  room  temperature,  and  of  feeble  strains. 
No  difference,  such  as  alleged  by  Mavrojannis,  exists  between  different 
species.     The    stage   of   liquefaction    in   which   formalin   produces 


Experiments  with  Bacterial  Enzymes  145 

hardening  is  simply  an  early  stage  of  digestion,  and  is  followed  under 
favorable  conditions  in  all  liquefying  species  by  a  state  of  permanent 
fluidity. 

10.  The  bacterial  hemolysins  and  bacterial  gelatinases  are  entirely 
distinct.  In  no  case  is  there  reason  to  believe  that  the  bacterial  gela- 
tinases can  produce  hemolysis. 


A   STATISTICAL  STUDY  OF  GENERIC  CHARACTERS   IN 

THE  COCCACEiE* 

C.-E.  A.  WiNSLOW  AND  Anne  F.  Rogers, 

ASSISTED   BY 

Elizabeth  Strongman,   Bertha  I.  Barker,  Mary  D.  Hale,  and 

Annie  P.  Hale. 

I.   Purpose  of  the  Investigation.  ' 

II.   Methods  of  the  Investigation. 

1.  Isolation  of  Cultures. 

2.  Selection  of  Characters  for  Study. 

3.  Morphological  Characters. 

4.  Cultural  Characteristics. 

5.  Biochemical  Reactions. 

III.  Results  of  the  Investigation. 

1.  Habitat. 

2.  Grouping  of  Cells,  and  Dimensions. 

3.  Gram  Stain. 

4.  Surface  Growth. 

5.  Fermentation  of  Carbohydrates. 

6.  Reduction  of  Nitrates. 

7.  Optimum  Temperature. 

8.  Chromogenesis. 

9.  Gelatin  Liquefaction. 

IV.  Conclusions  from  the  Investigation. 

1.  Foundation  of  Subfamilies  and  Genera  among  the  Cocci. 

2.  Systematic  Summary. 
V.    References. 

I.   PURPOSE  OF  THE  INVESTIGATION. 

There  has  been  placed  in  the  hands  of  the  biologist  within  the 
last  fev^  years  a  new  instrument  of  research  of  the  highest  value. 
This  is  the  statistical  method,  first  suggested  for  the  study  of  human 
characteristics  by  Quetelet  (1846),  specifically  applied  to  the  bio- 
logical problems  of  variation  and  heredity  by  Galton  (1889),  and 
extended  and  developed  in  detail  by  Pearson  and  his  pupils.  The 
most  important  papers  on  this  subject  may  be  found  in  the  files  of 
the  Philosophical  Transactions  0}  the  Royal  Society  0}  London  and 
in  Biometrika.  Admirable  brief  summaries  have  been  prepared 
by  Pearson  (1900)  and  Bigelow  (1904). 

*  Received  for  publication  April  3,  1906. 

146 


Generic  Characters  in  the  Coccaceae  147 

In  many  fields  of  science  the  statistical  method,  in  its  strict  sense, 
is  not  applicable.  Where  laboratory  experiments  may  be  made,  as 
in  most  fields  of  physics  and  chemistry,  a  comparatively  small 
array  of  data  obtained  under  perfectly  controlled  conditions  may 
permit  the  derivation  of  laws  of  relationship  without  extensive  statis- 
tical analysis.  The  same  thing  is  true  in  certain  fields  of 
biological  research.  As  soon,  however,  as  we  proceed  to  the  subtler 
problems  of  evolution,  it  becomes  necessary  to  accumulate  a  large 
number  of  observations  and  to  analyze  them  by  recognized  statis- 
tical methods.  These  methods  alone  have  brought  order  out  of 
chaos  in  anthropology  (Ripley,  1899).  They  have  laid  the  first 
foundation  for  a  real  science  of  mental  and  social  phenomena  (Thorn- 
dike,  1904;  Woods,  1906).  They  offer  the  most  promising  clue  for 
tracing  the  true  relationships  among  the  lower  forms  of  plant  and 
animal  life. 

As  we  have  elsewhere  pointed  out,  the  classification  of  the  bac- 
teria presents  peculiar  difficulties. 

Morphological  distinctions  are  so  slight  that  physiological  characters  must  ne- 
cessarily be  invoked  in  order  to  separate  and  classify  the  various  organisms,  and  these 
physiological  characters  are  often  variable.  Pathogenicity  may  be  taken  as  a  type 
of  those  powers  of  the  organism  which  are  easily  and  profoundly  modified  by  external 
conditions.  On  the  other  hand,  there  are  numerous  characters  which  appear  to  be 
extremely  constant.  Such  minute  differences  as  occur  in  the  resistance  of  different 
races  to  unfavorable  conditions  often  remain  unchanged  through  long  periods  of 
cultivation.  In  using  these  constant  characters  for  classification  we  are  met  by  another 
difficulty.  Though  constant,  the  differences  are  very  minute,  and  in  studying  a  number 
of  organisms  a  perfect  gradation  is  often  found  between  the  widest  e.xtremcs.  This 
is  exactly  what  should  be  expected  from  organisms  which  reproduce  only  by  asexual 
methods,  since  it  is  the  fusion  of  independent  cells  which  swamps  minor  differences 
producing  the  uniformity  of  species  among  higher  plants.  W'hh  asexual  reproduc- 
tion every  minute  variation  which  is  inheritable  must  persist  unchanged  until  some 
Other  chance  variation  occurs.  Each  such  variation  means  a  new  and  different  type  of 
bacterium. 

The  immense  number  of  generations  which  may  succeed  each  other  in  a  short 
space  of  time  makes  boundary  lines  as  shifting  as  they  would  become  among  the  higher 
plants  if  a  dozen  geological  epochs  were  considered  all  at  once. 

Since  ^"ith  unicellular  organisms  acquired  characters  may  probably  be  inherited 
in  a  higher  degree  than  with  other  forms,  existing  races  of  bacteria  will  be  markedly 
influenced  by  the  selective  effect  of  environmental  conditions,  and  must  bc^ar  the  impress 
of  their  recent  history. 

There  are,  therefore,  no  species  among  the  bacteria  in  quite  the  sense  in  which  we 
ordinarily  use  the  word — as  indicating  a  group  of  individuals  bound  together  by  a  num- 


148  C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 

ber  of  constant  characters  and  easily  identified  by  mutual  fertility.  From  one  point 
of  view  each  distinct  race  might  be  considered  a  species;  but  to  apply  a  name  for  every 
grade  of  difference  in  each  varying  character  would  be  impracticable;  and  such  names 
could  have  no  true  specific  value.  The  best  solution  of  the  difficulty  is  the  establish- 
ment of  certain  types  around  which  the  original  organisms  may  be  more  or  less  closely 
grouped;  but  it  must  be  clearly  recognized  that  the  groups  thus  formed  are  defined  by 
relation  to  the  type  at  their  center  and  are  not  sharply  marked  off  at  their  extremities 
from  the  other  groups  adjacent.' 

For  these  reasons  the  science  of  systematic  bacteriology  has 
remained  in  a  notably  undeveloped  state.  A  score  of  large  groups 
of  bacteria  have  been  more  or  less  satisfactorily  recognized  by  Fliigge 
(1896)  and  others.  Certain  of  these  groups,  like  the  aerobic  spore- 
formers,  the  colon  bacilli,  and  the  diphtheria  bacilli,  doubtless 
represent  true  natural  famihes  or  genera.  In  one  such  group,  that 
of  the  aerobic  spore-formers,  where  appreciable  morphological  dif- 
ferences exist,  the  species  and  varieties  have  been  carefully  worked 
out  by  Chester  (1904).  Far  too  many  specific  names  among  the 
bacteria  however,  mean  less  than  nothing.  The  incomplete  descrip- 
tion of  a  vast  number  of  identical  or  minutely  differing  forms  has  led 
to  a  confusion  quite  disheartening  to  the  student  of  such  systematic 
works  as  those  of  Migula  (1900)  and  Chester  (i  901).  Among  the 
Coccaceae  we  have  compared  the  published  descriptions  of  445 
species  and  found  evidence  for  only  31  distinct  types  (Winslow  and 
Rogers,  1905).  These  are  defined  mainly  by  arbitrary  combina- 
tions of  the  three  characters  of  acid  production,  chromogenesis,  and 
the  liquefaction  of  gelatin.  It  is  small  wonder  that  most  bacteri- 
ologists have  abandoned  any  attempt  at  a  natural  classification, 
and  have  sought  refuge  in  such  frankly  arbitrary  schematic  group- 
ings as  those  of  Fuller  and  Johnson  (1899),  Weston  and  Kendall 
(1902),  and  Jordan  (1903).  The  same  tendency  carried  to  its 
extreme  is  shown  in  the  decimal  systems  of  Gage  and  Phelps 
(1903),  and  Kendall  (1903),  and  in  the  modifications  recently  adopted 
by  the  Society  of  American  Bacteriologists. 

These  systems  are  most  valuable  for  a  routine  descriptive  work, 
and  for  arranging  and  cataloguing  records  of  cultures.  They  may, 
however,  lead  to  error,  unless  used  with  due  caution.  In  the  first 
place,  the  determinations  on  which  such  schemes  are  based  are  usually 
qualitative  only  and  not  quantitative.     In  the   second   place,  the 

'  Winslow  and  Rogers,  1905. 


Generic  Characters  in  the  Coccaceae  149 

application  to  all  bacteria  of  one  fixed  series  of  characters  arranged 
in  an  arbitrary  order  tends  to  suggest  a  mechanical  view  of  bacterial 
relationships  which  is  very  far  from  the  complex  truth. 

In  order  to  obtain  a  just  idea  of  the  real  relations  of  organisms, 
it  is  necessary  to  consider  each  systematic  group  by  itself.  As 
Robinson  has  pointed  out  in  an  admirable  paper  on  generic  classi- 
fication (Robinson,  1906),  "a  difference  having  great  classificatory 
significance  in  one  place  may  be  almost  valueless  in  another."  In 
studying  any  one  group  it  is  therefore  necessary  to  examine  afresh 
each  of  the  various  characters  used  for  the  identification  of  bac- 
teria in  general,  and  to  determine  its  loral  value  and  significance. 
Secondly,  under  each  character  it  is  necessar}^  to  determine  how 
many  distinct  types  of  structure  or  function  may  occur.  This  can 
be  done  only  by  measuring  the  character  quantitatively  in  a  large 
series  of  individuals,  and  plotting  curves  of  frequency  which  will 
show  whether  the  individual  forms  fluctuate  about  one  or  several 
modes.  This  has  been  attempted  by  Howe  (1904)  with  good  results, 
for  the  composition  of  the  gas  produced  in  dextrose  broth  by  organ- 
isms of  the  B.  coli  group. 

Finally,  the  correlation  between  various  properties  should  be 
determined,  since  it  is  obvious  that  the  presence  of  several  distinct 
characters  in  association  is  generally  of  more  significance  in  classi- 
fication than  that  of  any  one  alone. 

In  the  present  study  we  have  attempted  to  obtain  the  data  indi- 
cated, for  certain  groups  of  the  Coccaceae.  We  have  measured  the 
easily  and  definitely  measurable,  variable  characters  in  500  sepa- 
rately isolated  races  of  organisms,  and  analyzed  the  data  obtained, 
with  two  ends  in  view.  We  have  first  plotted  the  frequency  curve 
for  each  character  to  find  whether  the  array  varies  about  one  or  sev- 
eral modes,  and  where  the  modes  are  situated,  with  some  measure 
of  the  extent  of  variation  about  these  centers.  In  the  second  place, 
we  have  calculated  correlation  factors  for  the  most  significant  pairs 
of  characters.  Each  mode  on  the  curves  of  frequency  may  fairly 
be  taken  to  mark  a  natural  species  or  variety,  and  the  characters 
which  vary  together  must  form  the  most  important  basis  for  the 
establishment  of  the  larger  groups.  By  such  a  method  alone  it  is 
possible  to  locate  those  mountain  peaks  in  the  chain  of  bacterial 


150  C.-E.  A.  WiNSLOW  AND  Anne  F.  Rogers 

variations  which  rightly  deserve  generic  and  specific  names,  although 
records  of  the  characters  of  individual  races  by  the  decimal  system 
are  of  the  greatest  value  in  mapping  out  intermediate  regions.  Only 
the  statistical  study  of  numerous  individuals  by  comparable  quan- 
titative methods  can  reveal  the  general  laws  of  natural  classification 
among  the  bacteria;  and  this  study  must  be  made  in  each  group 
with  an  open  mind  free  from  arbitrary  predispositions. 

We  desire  in  advance  to  deprecate  a  comparison  between  the 
present  work  and  the  numerous  detailed  and  exact  biometrical  stud- 
ies which  have  appeared  in  other  fields.  In  bacteriology  our  methods 
of  measurement  are  crude  and  tedious,  and  the  general  knowledge 
requisite  for  the  selection  of  a  homogeneous  mass  of  material  is  lack- 
ing. We  should  know  the  outlines  of  the  general  groups  of  the 
cocci,  for  example,  before  we  can  properly  select  material  to  study 
variation  in  any  one  of  them. 

II.    METHODS  OF  THE  INVESTIGATION. 

I.  Isolation  of  Cultures. 

With  regard  to  the  larger  groups  of  the  Coccace^  we  have  else- 
where shown  (Winslow  and  Rogers,  1905)  that  the  family  could 
be  divided  into  two  subfamilies  and  five  genera,  defined  as  follows: 

Subfamily  i,  Paracoccaceae  (Winslow  and  Rogers):  Parasites 
(thriving  only,  or  best,  on,  or  in,  the  animal  body).  Thrive  well 
under  anaerobic  conditions.  Many  forms  fail  to  grow  on  artifi- 
cial media;  none  produce  abundant  surface  growths.  Planes  of 
fission  generally  parallel,  producing  pairs,  or  short  or  long  chains. 

Genus  i,  Diplococcus  (Weichselbaum) :  Strict  parasites.  Not 
growing,  or  growing  very  poorly,  on  artificial  media.  Cells  normally 
in  pairs  surrounded  by  a  capsule. 

Genus  2,  Streptococcus  (Billroth):  Parasites  (see  above).  Cells 
normally  in  short  or  long  chains  (under  unfavorable  cultural  con- 
ditions, sometimes  in  pairs  and  small  groups,  never  in  large  groups 
or  packets).  On  agar  streak  effused,  translucent  growth,  often 
with  isolated  colonies.  In  stab  culture  little  surface  growth.  Sugars 
fermented  with  formation  of  acid. 

Subfamily  2,  Metacoccaceae  (Winslow  and  Rogers):  Facultative 
parasites   or  saprophytes.     Thrive   best   under  aerobic   conditions. 


Generic  Characters  in  the  Coccaceae  151 

Grow  well  on  artificial  media,  producing  abundant  surface  growths. 
Planes  of  fission  often  at  right  angles;  cells  aggregated  in  groups, 
packets,  or  zooglca  masses. 

Genus  3,  Micrococcus  (Hallier)  Cohn:  Facultative  parasites  or 
saprophytes.  Cells  in  plates  or  irregular  masses  (never  in  long 
chains  or  packets).     Acid  production  variable. 

Genus  4,  Sarcina  (Goodsir) :  Saprophytes  or  facultative  para- 
sites. Division  under  favorable  conditions  in  three  places,  pro- 
ducing regular  packets.     Sugars  as  a  rule  not  fermented. 

Genus  5,  Ascococcus  (Cohn):  Generally  saprophytic  and  cells 
imbedded  in  large,  irregularly  lobed  masses  of  zooglea,  in  process 
of  carbohydrates.     Acid  usually  formed. 

In  the  present  investigation  we  have  included  representatives 
of  only  three  of  these  genera.  The  organisms  belonging  to  the  genus 
Diplococcus  do  not  lend  themselves  to  comparative  study  on  account 
of  the  difficulty  with  which  they  may  be  cultivated,  and  representa- 
tives of  the  genus  Ascococcus  occur,  if  at  all,  only  in  certain  peculiar 
habitats.  We  have  limited  our  study  to  forms  which  can  be  found 
in  ordinary  environments,  and  which  may  be  cultivated  on  ordinary 
laboratory  media;  that  is,  to  the  genera  Streptococcus,  Micrococcus, 
and  Sarcina. 

We  have  procured  our  cultures  in  approximately  equal  propor- 
tions from  five  different  sources:  from  the  internal  tissues  of  the  dis- 
eased human  body,  from  the  outer  surfaces  of  the  normal  human 
body,  from  water,  from  earth,  and  from  air.  Cultures  classed  under 
Habitat  I,  the  tissues  of  the  diseased  body,  were  obtained  chiefly 
from  the  Boston  City  Hospital,  and  the  Massachusetts  General 
Hospital,  of  Boston,  and  the  Johns  Hopkins  Hospital,  of  Baltimore. 
We  desire  to  express  our  cordial  thanks  to  the  bacteriologists  of 
these  institutions  for  their  courtesy  in  furnishing  us  with  these  organ- 
isms. The  cultures  classed  under  Habitat  II,  surfaces  of  the  normal 
body,  were  obtained  from  three  sources.  A  considerable  number 
were  isolated  from  serum  tubes,  received  by  the  Boston  Board  of 
Health  for  diphtheria  diagnosis.  In  this  connection  we  desire  to 
acknowledge  the  courtesy  of  the  bacteriologists  of  the  Board.  Only 
those  cultures  which  gave  a  negative  diagnosis  for  diphtheria  were 
used.     Another  series  of  cocci  was  isolated  from  the  hands  of  students 


152  C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 

in  the  Massachusetts  Institute  of  Technology.  In  collecting  them 
each  subject  rubbed  the  front  and  back  of  one  hand  with  a  wet  wad 
of  sterile  cotton,  running  the  wash  water  into  a  sterile  cup.  Finally 
a  small  number  of  cultures  were  obtained  from  excreta  of  man  and 
animals.  Under  Habitat  III  cultures  were  obtained  from  a  wide 
variety  of  natural  waters — public  supplies,  streams,  ponds,  pools, 
shallow  wells,  driven  wells,  and  the  sea.  Samples  were  taken  as 
far  as  possible  only  from  sources  held  to  be  free  from  pollution. 
Under  Habitat  IV  organisms  were  isolated  from  various  samples 
of  earth,  loam,  clay,  sand,  etc.,  obtained  mainly  in  different  regions 
of  eastern  Massachusetts.  The  cultures  grouped  under  Habitat  V 
were  taken  from  plates  exposed  to  the  air,  indoor  and  out,  and  here 
are  also  included  certain  organisms  of  unknown  origin  which  appeared 
as  contaminations,  or  for  whose  previous  history  we  have  no  record. 
In  each  case  the  sample  to  be  studied  was  first  plated  on  agar 
and  incubated  at  20°.  Colonies  which  looked  like  cocci  (not  pos- 
sessing, that  is,  the  characters  of  such  well-marked  forms  as  B. 
mesentericus,  B.  Zopfi,  or  B.  fluorescens)  were  fished  to  agar  streaks; 
from  each  sample  only  one  culture  was  taken,  unless  several  distinct 
types  of  colonies  appeared.  The  agar  streak  cultures  were  examined 
under  the  microscope  and,  if  apparently  cocci,  were  replated  in  order 
to  insure  their  purity,  again  transferred  to  agar  streaks,  and  again 
examined  under  the  microscope.  All  this  preliminary  work  was 
carried  out  at  20°,  and  the  stock  cultures  finally  obtained  were  kept 
on  agar  at  the  same  temperature.  There  can  be  no  doubt  that  by 
this  method  of  procedure  we  failed  to  obtain  many  of  the  more  strictly 
parasitic  streptococci  which  grew  only  feebly  on  solid  media  and 
are  most  active  at  a  temperature  of  37°.  This  fact  must  be  taken 
into  account  in  interpreting  our  results.  For  Micrococcus  and 
Sarcina,  however,  the  series  should  be  fairly  representative. 

2.  Selection  of  Characters  for  Study. 

The  characters  ordinarily  used  in  descriptive  bacteriology  are  few, 
particularly  in  a  group  of  such  simple  morphology  and  limited  bio- 
chemical powers  as  the  Coccaceae.  This  number  must  be  still  further 
reduced,  however,  when  we  come  to  inquire  which  of  them  really 
indicate  constant  and  independent  variations.     In  the  first  place,  it 


Generic  Characters  in  the  Coccaceae  153 

is  necessary  to  eliminate  properites  which  are  due  mainly  to  the  char- 
acter of  the  medium  and  the  conditions  of  incubation.  As  we  shall 
show  later,  those  minute  differences  in  the  appearance  of  colonies  on 
gelatin  which  form  the  basis  for  a  large  number  of  German  descrip- 
tions, fall  mainly  under  this  head.  Secondly,  many  characters,  while 
really  belonging  to  the  organism  itself  at  a  given  moment,  are  so 
easily  modified  by  cultivation  under  other  conditions  as  to  be  prac- 
tically worthless  in  systematic  work.  Among  the  cocci,  pathogenicity 
is  a  property  of  this  sort.  In  the  third  place,  it  is  evidently  unfair  to 
give  independent  weight  to  characters  which  are  simply  the  indirect 
result  of  other  properties  already  recorded.  Thus  among  the  cocci 
differences  in  broth  cultures  are  closely  connected  with  the  size  of  the 
cell  aggregates.  Organisms  growing  in  large  groups,  like  most  of 
the  sarcinae,  produce  heavy  sediment  and  often  colony-like  groups 
on  the  walls  of  the  tube,  while  those  in  which  the  cells  readily  sepa- 
rate exhibit  a  more  diffuse  turbidity.  Plate  cultures  add  little  more 
information  than  may  be  obtained  by  a  careful  scrutiny  of  stabs  and 
streaks;  and  the  growth  on  potato  and  blood  serum  in  many  groups 
of  bacteria,  and  particularly  among  the  cocci,  are  only  valuable  as 
measures  of  that  extremely  fugitive  quality,  the  general  vigor  of  the 
culture. 

The  considerations  which  have  influenced  us  in  the  selection 
of  characters  for  study  among  the  Coccaceae  may  be  conveniently 
arranged  in  the  order,  and  under  the  headings,  of  the  Report  of  the 
Committee  on  Standard  Methods  of  Water  Analysis  to  the  Labora- 
tory Section  of  the  American  Public  Health  Association  (1905). 

3.  Morphological  Characters. 

Form, — The  form  of  the  individual  cell  furnishes  no  help  in  the 
classification  of  the  Coccaceae,  since  under  favorable  conditions 
all  appear  as  regular  spheres.  Irregular  oval  forms  occur  at  times, 
particularly  in  cultures  freshly  isolated  from  the  throat  or  alimcntar}- 
tract,  but  the  form  usually  becomes  normal  after  cultivation. 

Manner  0}  grouping. — The  grouping  of  the  cell  elements  offers 
a  character  of  considerable  importance  among  these  bacteria.  While 
the  cocci  do  not  exhibit  an  entirely  unchanging  form  of  grouping, 
the  individuals  do  show  a  distinct  tendency  to  occur  in  one  of  four 
forms — either  in  pairs,  chains,  masses,  or  packets. 


154  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

The  grouping  is  somewhat  influenced  by  the  age  of  the  culture 
and  by  the  kind  of  medium  on  which  it  has  grown.  Even  the  same 
culture  will  show  wide  variation  from  the  typical  arrangement  of  the 
elements.  For  instance,  streptococci  occur  singly,  in  pairs,  chains, 
and  small  masses;  but  the  most  frequent  arrangement,  and  that  ob- 
tained under  the  most  favorable  conditions  (in  liquid  media),  is  in 
chains.  Again,  sarcinae  occur  singly,  in  pairs,  and  in  small  masses 
as  well  as  in  packets,  yet  the  typical  form  is  the  sarcina-packet. 
Cocci  grown  on  Nahrstoff  regularly  occur  in  plates,  and  usually  cap- 
sulated  ones. 

In  a  number  of  preliminary  studies  we  compared  the  groupings 
of  the  same  cultures  in  various  media  and  under  various  conditions, 
examining  cultures  of  different  ages,  from  nutrient  broth,  sugar 
broth,  peptone  solution,  hay  infusions,  nutrient  agar,  and  gelatin, 
and  acid  and  alkaline  gelatin.  Cultures  more  than  two  weeks  old 
showed  abnormalities  both  in  the  individual  cell  and  in  its  groupings. 
With  this  exception,  the  differences  produced  were  very  slight.  The 
only  constant  effect  of  the  medium  upon  grouping  which  was  apparent 
was  a  more  distinct  development  of  chains  in  liquid  cultures.  Organ- 
isms which  appear  as  long  chains  in  fresh  broth  cultures  may  show 
only  short  chains  with  irregular  groups  on  solid  media.  In  the  pres- 
ent study  we  have  omitted  the  broth  morphology  for  lack  of  time, 
and  have  recorded  the  grouping  only  as  apparent  on  the  agar  streak. 

The  streaks  used  were  never  more  than  three  days  old,  and  the 
grouping  was  observed  after  staining  lightly  with  methylene  blue  and 
mounting  in  cedar  oil.  Too  heavy  staining  may  introduce  a  serious 
error  by  making  packets  of  small  sarcinae  appear  like  large  single 
cells.  These  observations  on  the  culture  stained  with  methylene 
blue  were  controlled  by  careful  observations  of  the  slides  prepared 
for  the  study  of  the  Gram  stain,  as  noted  later. 

We  have  distinguished  two  main  groupings  only  by  this  method 
of  examination.  The  occurrence  of  packets  marks  one,  and  the  ab- 
sence of  packets  the  other,  group.  In  the  first  group  occur  the 
streptococci,  which  produce  pairs,  long  chains,  and  irregular  groups; 
and  the  micrococci,  which  show  pairs,  short  chains,  fours,  and 
irregular  groups;  while  the  sarcinse  include  organisms  which 
produce    fours,    irregular   groups,    and    packets,    as    well    as   those 


Generic  Characters  in  the  Coccaceae 


■?5 


extreme  forms  which  show  only  packets.  None  of  these  differences 
but  that  between  the  presence  and  absence  of  packets  appear  on 
agar  with  sufficient  constancy  to  be  determined  definitely.  For 
distinction  between  streptococci  and  micrococci  the  observation 
of  broth  cultures  would  perhaps  be  valuable. 

Dimensions. — The  cocci  exhibit  a  range  in  size  from  o .  i  to 
2.0  /i  with  considerable  variation  between  individual  cells  in  the 
same  culture.  We  were  somewhat  surprised  to  find  that  we  could 
demonstrate  no  definite  relation  between  size  and  the  age  of  cul- 
tures, or  the  conditions  of  cultivation.  In  a  series  of  preliminary 
studies  the  same  organism  was  grown  on  seven  kinds  of  media  and 
examined  at  intervals  during  a  period  of  two  months.     The  maxi- 

120 


100 


JZA 


80 
60 
40 
20 


.1         .2        .5        .4.        .5        .6        .7        .8        .9        10 

Fio.  I.— Dimensions  of  345  cocci.     Abscissae,  average  diameter  in  i^.    Ordinates,  number  of  cultures. 

mum  size,  in  different  cultures,  was  recorded  on  the  first,  second, 
seventh,  14th,  42d  days,  and  after  two  months  respectively.  The  maxi- 
mum size  developing  in  the  different  kinds  of  media  during  those 
two  months  was  found,  respectively,  in  broth  at  37°,  broth  at  20°, 
Nahrstoff-Heyden,  nutrient  gelatin,  acid  and  alkaline  gelatin,  and 
under  anaerobic  conditions.  In  other  words,  the  age  and  kind  of 
medium  had  no  constant  effect,  except  that  in  most  cases  the  Nahr 
stoff  and  other  poor  media  showed  the  smallest  individuals.     No 


'// 


156  C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 

constant  difference  in  size  was  apparent  in  comparing  solid  and  li- 
quid cultures.  One  series  of  organisms  examined  in  dextrose  broth 
and  on  agar,  at  periods  ranging  from  one  day  to  two  weeks,  showed 
the  same  average  size  in  both  media  and  at  all  ages.  Finally  we 
attempted  to  see  whether  prolonged  cultivation  under  special  con- 
ditions would  affect  the  size  of  the  cell.  Cultures  were  grov^n  for 
10  days,  in  broth  at  37°,  on  nutrient  gelatin,  and  on  acid  and 
anaerobic  gelatin  with  daily  transfers.  The  size  of  each  culture 
was  recorded  on  the  loth  day,  after  which  time  each  was  transferred 
to  gelatin  and  examined  after  one  day.  The  results  showed  prac- 
tically no  significant  differences. 

In  a  comparison  of  the  size  as  determined  by  examination  of  living 
organisms  and  of  stained  preparations,  the  cells  appeared  generally 
somewhat  smaller  after  staining.  This  is  no  doubt  partly  due  to  some 
shrinkage  in  drying,  and  partly  to  the  imperfect  definition  which 
makes  the  unstained  specimens  appear  larger  than  they  really  are. 
Occasionally,  when  the  staining  was  too  heavy,  the  stained  cells 
appeared  larger.  In  any  case  the  differences  are  unimportant, 
and  we  have  used  the  size  of  the  mcthylene-blue-stained  preparation 
throughout  our  work. 

Staining  reactions.  —  Since  the  cocci,  as  far  as  we  have  examined 
them,  all  stain  easily  with  methylene  blue,  we  have  made  no  special 
tests  with  anilin-gentian-violet.  The  Gram  stain  has,  however,  been 
used  on  all  our  cultures,  since,  in  the  genus  Diplococcus  and  in  many 
other  groups,  it  has  been  thought  to  have  such  special  importance. 

The  value  of  this  staining  method  has  been  studied  with  consid- 
erable care  by  Mr.  A.  T.  Brant,  working  in  the  laboratories  of  the 
Institute.  Mr.  Brant  found,  as  other  observers  have  done,  that  while 
certain  bacteria  are  constantly  Gram-negative  or  Gram-positive, 
others  exhibit  an  intermediate  condition,  retaining  the  stain  under 
some  conditions  and  giving  it  up  under  others.  In  his,  as  yet  unpub- 
lished, paper  he  notes,  for  example,  that  all  cultures  of  B.  coli  are 
decolorized  by  one  minute's  treatment  with  alcohol,  while  B.  mega- 
therium constantly  fails  to  decolorize  after  three  hours.  On  the 
other  hand,  with  B.  fluorescens,  M.  pyogenes,  M.  aureus,  and  B. 
diphtheriae  the  result  is  affected  by  the  time  of  decolorization,  as  well 
as  by  the  age   of  the   cultures.     Between  the  fixed  points  at   the 


Generic  Characters  in  the  Coccaceae  157 

extreme,  preparations  will  yield  varying  results,  showing  some  cells 
stained  and  others  decolorized.  As  a  rule,  the  large  majority  of 
cells  in  a  given  preparation  will  show  one  reaction  or  the  other; 
but  a  second  slide  made  from  a  similar  doubtful  case  might  yield 
a  different  result. 

The  time  chosen  for  decolorization  is,  of  course,  an  arbitrar}' 
factor  which  will  affect  the  proportion  of  positive  results  obtained. 
In  our  work,  as  a  result  of  Mr.  Brant's  experiments,  we  fixed  on 
three  minutes,  although  we  are  not  certain  that  this  is  really  pref- 
erable to  the  five-minute  period  fixed  by  the  Committee  on  Standard 
Methods.  We  have  applied  the  anilin-oil-gentian-violet  for  one 
and  a  half  minutes,  and  the  Gram  solution  for  one  and  a  half  minutes 
instead  of  the  one-  and  two-minute  periods  of  the  committee. 

In  all  cases  we  made  the  stain  on  young  20°  agar  cultures  (not  over 
five  days  old),  and  in  each  case  the  test  was  made  in  duplicate  at 
different  times.  When  the  results  of  the  two  tests  coincided,  the  culture 
was  recorded  as  positive  or  negative.  Cultures  which  gave  one  posi- 
tive and  one  negative  test,  or  in  which  the  stained  and  decolorized 
appeared  in  about  equal  proportions,  are  recorded  in  an  intermediate 
class. 

Flagella. — As  a  result  of  the  work  of  Ellis  (1902),  we  have  devoted 
considerable  time  to  the  study  of  motility  among  the  cocci.  This 
author  reported  the  finding  of  spores  and  flagella  in  various  strepto- 
cocci and  sarcinaj,  and  Arthur  Meyer  carried  this  position  to  an 
extreme  in  the  statement  that  "  the  researches  of  Ellis  have  rendered 
it  doubtful  whether  there  are  any  species  of  bacteria  which  entirely 
lack  flagella"  (Meyer,  1903).  We  examined  a  number  of  cultures 
very  carefully,  transferring  them  at  frequent  intervals  on  different 
media,  according  to  the  general  plan  adopted  by  Ellis.  We  found 
in  almost  every  case  active  vibratory  movements,  with  a  tendency 
to  incomplete  rotation,  the  successive  jerks  sometimes  producing  a 
gradual  translation  across  the  field.  This  type  of  behavior  is 
entirely  different  from  the  true  motility  characterized  by  slow,  steady 
revolution,  which  appears  in  such  forms  as  S.  agilis.  We  are  con- 
vinced that  most  of  the  cocci  are  non-motile,  while  a  few  forms  show 
true  movement;  it  is  with  this  type  of  motility  that  clearly  stainable 
flagella  have  been  found  associated.     The  study  of  this  character  is 


158  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

therefore  of  significance.  It  is  questionable,  however,  whether  it  is 
one  of  the  most  important  characters  in  this  group  of  bacteria.  It 
appears  from  the  published  descriptions  of  species  that  this  property 
is  not  correlated  with  any  other  character,  arising  independently  in 
forms  exactly  resembling  non-motile  forms  in  every  other  respect. 
On  account  of  its  rarity  and  this  apparent  lack  of  correlation  with 
other  differences,  as  well  as  on  account  of  the  difficulty  of  studying  it, 
the  property  of  motility  has  been  so  far  omitted  from  the  present  study. 

Spores. — The  experiments  carried  out  by  Ellis  (1902)  strongly 
suggest  the  presence  of  specially  resistant  cells  in  old  cultures  of 
the  cocci.  His  figures  are,  however,  by  no  means  conclusive  as 
to  the  existence  of  true  spores.  In  the  absence  of  any  observations 
as  to  germination,  we  have  not  felt  that  the  evidence  warranted 
extensive  microscopic  study  of  this  character. 

Fission. — A  study  of  the  conditions  influencing  the  growth- 
forms  of  the  Coccaceae  should  be  of  considerable  interest.  Pairs 
and  chains  are  apparently  associated  with  meager,  and  groups  and 
packets  with  more  abundant,  development.  The  effect  of  the  gen- 
eral rate  of  growth  must,  however,  be  modified  by  the  rate  at  which 
cell-wall  and  cell-protoplasm,  respectively,  are  formed. 

A  careful  study  of  the  method  by  which  these  groupings  arise 
in  cell-division,  such  as  could  be  made  by  the  use  of  Hill's  hanging- 
block  method,  would  no  doubt  throw  much  light  on  all  such  points, 
and  should  precede  any  final  conclusions  as  to  the  relationships  of 
the  cocci.  In  examining  a  large  number  of  organisms,  however,, 
the  agar  block  would  have  proved  too  time-consuming.  We  have 
therefore  hmited  ourselves  to  the  observations  made  on  stained 
preparations  from  ordinary  cultures. 

Capsules. —  Considerable  preliminary  work  failed  to  indicate  any 
constant  differences  in  capsule  formation  among  the  cocci  studied. 
This  character  appears  to  be  of  considerable  value  among  the 
diplococci  (Buerger,  1904);  but  even  with  them  it  varies  markedly 
with  the  medium  used  for  cultivation.  We  cultivated  certain  select- 
ed organisms  in  broth  at  20°  and  at  37°,  on  nutrient  gelatin,  acid 
gelatin,  alkaline  gelatin,  anaerobic  gelatin,  and  Nahrstoff-Heyden 
agar,  and  examined  them  at  intervals  by  Welch's  staining  method. 
In  every  case  capsules  were  apparent  at  some  stages,    being   most 


Generic  Characters  in  the  Coccaceae  159 

strongly  developed  in  old  cultures  and  on  poor  media  like  the 
Nahrstoff  agar.  This  character  has  not  seemed  to  us  of  suflicient 
diagnostic  value  to  be  included  in  our  routine  examinations. 

Involution  and  degeneration  jorms. — In  numerous  examinations 
of  old  cultures  we  found  no  involution  forms  of  special  significance. 
As  noted  above,  swollen  and  oval  forms  are  more  apt  to  occur  in  old 
cultures  of  cocci,  but  they  are  not  sufficiently  definite  to  warrant 
record. 

4.  Cultural  Characters. 

In  a  study  of  this  sort  we  have  necessarily  included  only  those 
tests  which  reveal  definite  and  independent  variable  characters. 
Most  of  the  commonly  observed  cultural  characteristics  are  the  sec- 
ondary results  of  a  few  fundamental  properties  which  can  be  observed 
on  one  medium  as  well  as  on  several.  For  this  reason  we  have  elimi- 
nated a  number  of  the  ordinary  media  from  our  routine.  The  general 
character  of  the  growth  is  approximately  the  same  on  agar,  blood 
serum,  potato  or  Nahrstoff,  except  that  agar  has  always  markedly 
more  growth  and  potato  often  none.  An  organism  producing 
abundant  chromogenic  growth  on  agar  will  give  good  growth  and 
some  pigment  on  the  other  media.  The  streptococcus  growth  (>n 
agar  gives  restricted  and  veil-like  growth  on  serum  and  Nahrst^  tf, 
and  usually  no  growth  on  potato.  In  other  words,  NahrstolT  agar, 
serum,  and  potato  are  simply  poorer  media  than  agar,  and  sh'nv  no 
specific  characteristics  other  than  those  due  to  feebleness  of  growth. 
Blood  serum  may  be  useful  in  other  groups  to  show  a  special  type 
of  liquefaction,  but  in  a  preliminary  study  of  50  of  our  cultures  we 
never  found  this  to  occur,  and  it  is  nowhere  recorded  in  pubhshed 
descriptions  of  the  Coccaceae.  In  25  out  of  50  cultures  grown  on 
potato  no  growth  occurred,  and  in  no  case  have  we  observed  dis- 
coloration. These  media  have  therefore  been  omitted.  This  action 
is  in  accordance  with  the  conclusions  of  the  Committee  on  Standard 
Methods  (1905),  in  considering  their  value  for  general  diagnostic  use. 

Nutrient  broth. — In  the  group  of  the  cocci  we  have  not  found 
that  any  information  of  definite  value  could  be  derived  from  a  study 
of  broth  cultures.  None  of  the  forms  studied  form  a  surface  pel- 
licle or  produce  any  characteristic  odor.  There  remain  to  be  ob- 
served only   two   features — turbidity   and   sediment — which   in   our 


i6o 


C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 


judgment  depend  directly  on  other  properties,  such  as  the  general 
vigor  of  growth  and  the  size  of  the  cell  aggregates.  Both  turbidity 
and  sediment  vary  markedly  with  the  age  of  the  culture;  what  is 
first  turbidity  later  settles  to  form  sediment,  as  the  waste  products 
of  the  bacteria  check  their  development.  The  amount  of  either 
depends  on  the  activity  of  growth.  A  constant  difference  often  appears 
between  cultures  which  early  in  the  course  of  development  show 
considerable  turbidity  with  little  or  no  sediment,  and  those  which 
almost  at  once  develop  a  heavy  sediment  v/ith  colony-like  masses 


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Fig.  2. — Acid  production  of   500    cocci;   in   dextrose   broth 


and  lactose   broth  = 


AbscissaE^  acidity  in  per  cent  normal.     Ordinates,  number  of  cultures. 


of  growth  cHnging  to  the  walls  of  the  tube.  This  difference,  how- 
ever, appears  to  be  correlated  with  the  growth-form  and  general 
vigor  of  the  coccus.  Organisms  of  the  Streptococcus  type  with  cells 
separating  readily,  which  show  faint  surface  growth,  produce  chiefly 
turbidity;  while  organisms  like  Sarcina  with  large  cell  aggregates 
and  rich  surface  growths,  show  hea\y  sediment. 

Gelatin    plates. — Minute    differences    in    the    macroscopic    and 
microscopic    appearance    of    colonies    on    gelatin    are   given    great 


Generic  Characters  in  the  Coccaceae  i6i 

weight  in  German  systems  of  classification.     Certain  special  char- 
acteristics do,  indeed,  appear  in  old  gelatin  colonies  of  the  cocci 
after  several   weeks   of   incubation.     Colonies   may   remain   almost 
spherical;  or  they  may  expand  in  fiat,  disclike  growths  with  terraced 
edges.     Sometimes  a  distinct  boss  appears  at  the  center,  surrounded 
by  a  flatter  area.     The  edges  may  be  entire,  or  more  or  less  deeply 
scalloped,  and  the  edges  of  the  scallops  may  be  produced  inward  in 
folds.   Concentric  rings  sometimes  appear  in  the  interior  of  the  colony, 
or  zones  of  partially  liquefied  gelatin  around  its  periphery.     Some 
of  these  characters  vary  without  any  apparent  reason,  as  different 
colonies  on  a  plate  show  different  characteristics;  this  is  perhaps 
due  to  differences  in  the  position  of  the  original  cell  relative  to  the 
gelatin  surface.     Most  of  them  are  profoundly  modified  by  variations 
in  the  amount  of  moisture  in  the  gelatin  and  in  the  atmosphere 
above.     In  a  series  of  comparative  studies  with  different  conditions 
of  incubation  we  found  that  highly  characteristic  colonies  of  granular 
structure,    with   deeply   lobed   edges   and   indented   surfaces,   could 
be  produced  by  cultivation  in  an  incubator  whose  atmosphere  was 
kept  dry  by  calcium   chloride.     Dunham    (1903)   has   pointed  out 
the  wide  differences  which  may  be  due  to  slight  variations  in  the 
physical  properties  of  the  gelatin  used.     Those  ditTerences  which 
are  really  characteristic  of  the  organisms  themselves  appear  to  be 
related  to  two  fundamental  powers :    the  general  vigor  of  growth  and 
the  liquefying  power.     It  may  be  possible  that  other  differences  exist 
in  old  gelatin  colonies  which  are  really  characteristic,  but    in    the 
present    state    of    knowledge    it    seemed    best    to    omit    the    gelatin 
plate  in  favor  of  more  definite  tests.     Liquefying  power  and  general 
vigor  of  growth  are  observed  in  the  gelatin  stab  and  the  agar  streak 
respectively. 

Gelatin  tubes. — All  our  cultures  have  been  studied  in  the  gelatin 
tube,  but  only  the  single  character  of  the  amount  of  liquefaction 
has  been  systematically  recorded.  The  distinction  between  difTerent 
non-liquefying  colonies  lies  in  the  amount  of  surface  growth  and  the 
color,  both  of  which  characters  are  more  easily  studied  on  the  agar 
streak.  The  character  of  the  surface  growth,  Hke  that  of  the  gelatin 
plate  colony,  does  not  appear  in  this  group  to  offer  any  character 
of  diagnostic  value,  and  all  the  cocci  grow  fairly  well  in  the  stab. 


i62  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

Among  the  liquefying  forms  we  have  not  found  the  shape  of  the 
liquefaction  of  sufficient  constancy  to  be  recorded.  Whipple  (1902) 
has  strikingly  shown  the  uncertainty  of  this  character — almost  every 
possible  type  appearing  in  media  made  with  slightly  different  com- 
mercial gelatins.  The  Committee  on  Standard  Methods  (1905)  has 
also  omitted  this  property. 

The  amount  of  liquefaction  of  gelatin  was  therefore  the  only 
character  recorded  on  the  gelatin  stab.  The  method  by  which  this 
was  measured  will  be  described  under  "Biochemical  Reactions." 

Agar  plates. — The  same  reasons  which  led  us  to  omit  the  gelatin 
plate  militate  against  the  use  of  the  agar  plate  as  a  diagnostic  test. 
Constant  differences  which  exist  between  colonies  are  slight  and 
depend  on  a  few  fundamental  properties  which  may  be  more  easily 
observed  on  other  media,  notably  on  the  agar  streak. 

Agar  tubes. — The  general  conclusion  from  what  has  been  said 
in  this  discussion  of  cultural  characteristics  is  that  in  the  cocci  a 
single  medium  is  sufficient  for  their  determination.  We  should, 
however,  deprecate  any  extension  of  thiS"  conclusion  to  other  groups 
where  the  gelatin  stab  or  the  plate  culture  may  yield  information  of 
definite  value.  Even  among  the  cocci  further  study  may  show  con- 
stant and  characteristic  differences  in  gelatin  colonies,  and  if  this 
should  be  the  case,  no  one  could  fail  to  welcome  an  addition  to  the 
meager  list  of  diagnostic  characters  at  our  disposal.  In  the  absence 
of  evidence  as  to  the  value  of  these  media,  we  feel  it  unwise  to  repeat 
tests  mechanically  and  without  any  definite  purpose,  merely  because 
they  have  had  an  important  place  in  the  historical  development 
of  the  science. 

All  cultural  characteristics  have  therefore  been  observed  in  the 
agar  tube.  A  combined  streak  and  stab  was  made  on  a  slant  sur- 
face, and  the  cultures  were  uniformly  studied  after  incubation  for 
two  weeks  at  20°  C.  Cultures  of  different  age  exhibit  marked  differ- 
ences, but  the  characters  of  the  old  cultures  are  the  outcome  of  those 
of  the  new.  Comparative  studies  with  lactose  agar  and  glycerin 
agar  showed  neither  to  be  as  favorable  a  medium  as  ordinary  nutrient 
agar. 

In  order  to  obtain  a  comparative  idea  of  cultural  characters 
we  examined  two  weeks'  agar  streaks  of  the  whole  500  cultures 


Generic  Characters  in  the  Coccaceae 


163 


at  the  same  time.  We  are  somewhat  surprised  to  lind  that  the  vis- 
ible differences  between  the  cuhures  were  due  almost  wholly  to  two 
properties — chromogenesis  and  the  general  vigor  of  surface  growth. 
There  was  a  distinction  in  luster  between  a  large  majority  of  the 


1 

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I 


n     Ri     IV 


VI     VD     Yin     IX 


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Fig.  3. — Distribution  of  500  cocci  according  to  chromogenesis.     Roman  numerals,  hues;    Arabic 
numerals,  chromos. 

cultures  w^hich  had  smooth  and  shining  surfaces,  and  a  few  which 
were  dull  and  rough.  This  difference  appears,  however,  to  be  due 
simply  to  the  relative  amount  of  growth  and  moisture  in  the  tube. 
Faint  growths  are  moist  and  shining,  while  heavy  growths  in  tubes 
which  do  not  contain  much  moisture  show  the  dry,  rough,  dull 
appearance.  The  "white  chromogens"  showed  another  slight 
difference,  varying  from  an  opaque  porcelain  white  to  a  duller  and 


164  C.-E.  A.  WiNSLOw  AND  Anne  F,  Rogers 

more  translucent  growth  of  indefinite  color  and  somewhat  shiny 
appearance;  but  there  was  no  sharp  boundary  to  separate  the  types. 
For  the  present  we  have  omitted  this  character,  akhough  it  may  prove 
to  be  of  importance  in  more  detailed  work. 

We  have  therefore  noted,  as  cultural  characters  on  the  agar  streak, 
only  the  color  production  and  the  vigor  of  surface  growth.  The 
method  of  studying  the  former  character  will  be  described  under 
" Chromogenesis."  Under  "Vigor  of  Surface  Growth"  we  found 
it  possible  to  distinguish  five  different  types.  Grade  i  includes 
forms  of  the  Streptococcus  type  which  form  only  a  very  faint,  veil- 
like, growth,  or  a  few  translucent  dotted  colonies  on  the  surface. 
Grade  2  is  reserved  for  a  somewhat  more  abundant,  but  still  meager, 
growth.  Grade  3  corresponds  to  a  good,  but  not  abundant,  streak; 
Grade  4,  to  an  abundant  growth;  and  Grade  5,  to  a  very  heavy 
surface  development. 

The  relation  to  free  oxygen  is  distinctly  involved  in  the  vigor 
of  surface  growth,  and  the  agar  streak  also  served  for  the  study  of 
various  other  biochemical  reactions.  Inhibition  of  growth  by  acid- 
ity and  alkalinity  of  media,  temperature  relations,  and  pigment  for- 
mation were  all  recorded  on  this  medium  under  conditions  to 
be  described  below. 

5.  Biochemical  Reactions. 

Action  upon  milk. — Milk  is  a  favorable  nutrient  medium  for  bacte- 
rial growth  because  of  its  rich  food  properties,  and  in  many  groups 
it  gives  important  information,  but  it  has  no  specific  diagnostic 
value  for  the  Coccaceae,  as  all  the  changes  it  undergoes  are  corre- 
lated with  those  which  occur  in  sugar  broths  and  with  the  general 
activity  of  the  organism.  No  coagulating  enzymes  and  casein- 
digesting  enzymes  are  found  in  this  group,  so  far  as  we  are  aware, 
and  no  gas  or  odor  is  produced.  The  only  changes  which  the  cocci 
effect  in  milk  are  therefore  the  production  of  acid  or  alkali,  coagu- 
lation and  decolorization  of  the  litmus. 

Decolorization  has  no  significance,  except  that  it  indicates  the  gene- 
ral activity  of  the  organism.  When  the  organism  is  most  active,  it  uses 
up  the  oxygen  and  reduces  the  litmus,  which  is  accordingly  decol- 
orized, and,  conversely,  when  activity  grows  less,  oxygen  diffuses 
from  the  surface  making  the  litmus  pink  again. 


Generic  Characters  in  the  Coccaceae  165 

Coagulation  depends  upon  the  amount  of  acid  produced,  and 
is  more  easily  studied  in  sugar-broth  cultures. 

Action  upon  carbohydrates. — The  characteristics  usually  observed 
in  sugar  broth  are  turbidity  and  sediment,  relation  to  oxygen,  gas 
production,  and  acid  production.  We  have  given  reasons,  in  dis- 
cussing nutrient  broth,  for  considering  turbidity  and  sediment 
unimportant,  and  the  relation  to  oxygen  is  most  sharply  defined 
by  surface  growth  in  the  agar  tube.  None  of  the  cocci,  so  far  as 
known,  produce  gas,  and  there  remains  only  acid  production  to 
be  recorded.  For  this  purpose  ordinary  straight  tubes  were  used. 
The  sugars  tested  were  dextrose  and  lactose.  Saccharose  has  been 
omitted  for  the  present,  for  lack  of  time.  A  preliminary  test  indi- 
cated that  this  sugar  is  less  commonly  fermented  by  the  cocci  than 
are  dextrose  and  lactose. 

The  media  were  made  up  in  the  usual  manner  with  2  per  cent 
of  the  sugar  to  be  tested.  The  reaction  was  made  about  neutral, 
and  after  tubing  and  sterilization  it  was  usually  between  0.5  and 
i.o  per  cent.  After  standing  for  two  weeks  sterile  blanks  showed 
a  slight  further  rise  in  acidity,  so  control  tubes  were  always  kept 
with  each  batch  inoculated  and  titrated  at  the  end  of  the  experiment. 
After  considerable  preliminary  experimentation,  it  was  decided 
to  titrate  with  phenolphthalein  as  an  indicator  in  the  cold.  Methyl 
orange  is  not  sensitive  to  the  organic  acids  and  gives  a  poor  end- 
point.  With  phenolphthalein  a  comparative  scries  of  titrations 
made  on  the  same  tubes,  first  cold  and  then  boiling,  showed  slightly 
higher  results  by  the  latter  method.  Evidently  heating  increases 
the  apparent  acidity  more  by  the  breaking-up  of  unstable  com- 
pounds than  it  decreases  it  by  driving  off  carbon  dioxide.  The 
cold  method  was  therefore  used.  To  5  c.c.  of  the  sugar  broth, 
grown  for  two  weeks  at  20°,  was  added  95°  c.c.  of  distilled  water 
and  two  or  three  drops  of  phenolphthalein.     This  was  titrated  against 

_  NaOH  and  from  the  value  obtained  was  subtracted  the  acidity 

30 

of  the  blank  controls  titrated  at  the  same  time.  All  tests  were  made 
in  duplicate,  and  the  final  value  recorded  as  the  acid  or  alkali  pro- 
duction of  the  organism  is  the  difference  between  tiic  average  of 
two  titrations  of  tubes  in  which   it    had   grown  for  two  weeks  and 


1 66 


C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 


the  average  of  two  blank  controls.  No  determination  was  made  of 
the  rate  of  acid  production  as  distinguished  from  this  total  final 
acidity,  though  such  observations  might  be  of  much  interest. 

Action  upon  nitrates. — Data  with  regard  to  the  reduction  of  nitrates 
by  the  cocci  are  extremely  meager,  the  presence  or  absence  of  this 
character  being  recorded  in  very  few  of  the  published  descrip- 
tions. It  seems,  however,  to  have  a  fair  degree  of  definiteness, 
and  we  have  included  it  as  a  qualitative  test  in  our  routine. 
Each  organism  was  inoculated  into  a  series  of  lo  tubes  of  standard 
nitrate  solution.  After  seven  days'  growth  at  20°  the  tubes  were 
tested  for  nitrites  and  ammonia  by  the  regular  method  prescribed 
by  the  Committee  on  Standard  Methods  (1905).  The  test  for  nitrates 
was  omitted  after  it  was  found  that  all  the  cultures,  out  of  a  con- 
siderable series  tested,  gave  positive  results,  without  exception. 
The  results  of  the  tests  for  nitrites  and  ammonia  are  expressed  in 
the  number  of  tubes  which  gave  positive  results,  out  of  the  10  which 
were  tested.  In  view  of  the  fair  constancy  of  the  reaction  as  observed, 
we  regret  that  this  test  was  not  made  quantitative. 

Production  oj  indol. — A  preliminary  examination  of  some  50  cul- 
tures showed  no  production  of  indol  in  any  case,  and  a  study  of  the 
literature  of  the  cocci  indicates  that  this  property  is  very  rare,  if  it 
ever  occurs,  in  this  group.  It  was  therefore  omitted  from  our 
routine. 

Inhibition  oj  growth  by  acidity  and  alkalinity  0}  media. — This 
character  is  of  considerable  importance  and  warrants  careful  study, 
but  it  is  obviously  a  difficult  property  to  observe  in  a  large  series  of 
cultures,  and  we  have  not  attempted  to  use  it  in  the  present  investi- 
gation. A  preliminary  examination  of  t,t,  cultures,  the  results  of 
which  are  shown  in  the  table,  indicated  that  i  per  cent  is  the  opti- 
mum acidity  for  a  majority  of  these  organisms,  and  that  an  excess 
of  acidity  over  this  amount  is  more  generally  fatal  than  an  alkahne 
medium. 

Optimum  Reaction  for  Growth  and  Color  Proddction. 
number  of  organisms. 


Optimum  Reaction 

—  1.0 

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0 

+  .5 

+  1.0 

+  1-5 

+  2.0 

Growth 

Color 

2 
3 

4 
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6 

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3 

Generic  Characters  in  the  Coccaceae  167 

Relation  to  free  oxygen. — The  Committee  on  Standard  Methods 
(1905)  recommends  that  the  relation  of  bacteria  to  oxygen  be  studied 
by  the  comparison  of  cuhures  made  under  normal,  and  under  anae- 
robic, conditions.  A  preliminary  study  of  50  cultures  made  in  this 
way  led  to  the  belief  that  such  a  procedure  is  unnecessary  among 
the  cocci.  All  but  two  of  the  cultures  studied  showed  some  growth 
under  anaerobic  conditions,  but  the  growth  was  in  most  cases  meager. 
It  became  evident  that  there  are  two  main  types  of  organisms:  those 
which,  like  Streptococcus,  grow  only  feebly  on  the  surface  of  aerobic 
agar,  and  which  grow  equally  well  under  anaerobic  conditions;  and 
those,  like  Sarcina,  which  form  abundant  surface  growths  under 
aerobic  conditions,  and  under  anaerobic  conditions  grow  feebly 
like  Streptococci.  In  other  words,  there  is  little  difference  between 
the  anaerobic  cultures  of  the  cocci.  Therefore,  for  purposes  of  classi" 
fication  we  have  considered  the  study  of  the  aerobic  surface  growth 
a  sufficient  measure  of  the  relation  to  ^ree  oxygen,  as  well  as  of 
general  vigor.  The  five  grades  recorded  under  vigor  of  surface 
growth  correspond  fairly  well  to  four  grades  of  aerobiosis,  from 
forms  anaerobic  and  facultatively  aerobic,  to  forms  which  are  strong 
aerobes. 

Temperature  relations. — There  are  two  points  of  special  importance 
which  ought  to  be  determined  in  studying  temperature  relations, 
the  optimum  temperature  and  the  high  death-point.  The  death- 
point  at  extremely  low  temperatures  is  too  indefinite  to  be  attempted, 
and  the  extreme  limits  of  growth,  although  desirable  data  may  be 
omitted  as  less  important  than  the  other  two  properties. 

For  the  determination  of  the  optimum  temperature  we  first  made 
a  series  of  preliminary  studies  by  comparing  agar  cultures  grown 
at  10°,  20°,  37°,  45°,  and  56°.  We  found  two  cultures  growing  bet- 
ter at  20°,  18  developed  equally  well  at  20°  and  37°,  22  showed  an 
optimum  at  37°,  two  grew  equally  well  at  37°  and  45°,  and  four  grew 
best  at  45°.  These  conclusions  refer  only  to  the  amount  of  growth, 
color  production  being  in  most  cases  most  active  at  20°.  From 
these  results  we  concluded  that  the  information  to  be  gained  by  cul- 
tures grown  below  20°  and  above  37°  would  be  scarcely  commensu- 
rate with  the  labor  involved,  and  we  have  limited  our  observation 
to  the  comparison  of  growth  and  color  production  at  20°  and  37°. 


1 68 


C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 


/  >>^' 
y 


Cultures  were  grown  for  this  purpose  on  agar  at  20°  for  two  weeks 
and  compared  by  inspection.  Amount  of  growth  and  depth  of 
color  were  recorded  in  five  arbitrary  grades  as  follows:  growth  or 
color  production,  much  better  at  20°,  somewhat  better  at  20°,  equal 
at  the  two  temperatures,  somewhat  better  at  37°,  and  much  better 
at  37°. 

Thermal  death-points  were  included  in  the  original  plan  of  our 
experiments  and  have  now  been  made  on  87  cultures.  The  process 
used  is  to  inoculate  from  three-  to  five-day-old  agar  cultures  into 
broth  tubes  brought  to  the  desired  temperature  in  a  water-bath 
heated  electrically  by  a  platinum  coil,  and  to  expose  them  for  10  min- 
utes. The  tubes  are  then  cooled  and  incubated  at  37°  for  six  days. 
At  the  end  of  that  time,  streaks  are  inoculated  from  the  broth  tubes 
in  order  to  make  sure  by  characteristic  growth  that  the  organisms 
orginally  inoculated  are  present.  Tests  are  made  from  55°  up  to 
the  point  where  growth  fails.  The  process  is  so  tedious  that  we  have 
been  unable  to  complete  the  work,  and  must  omit  this  property  for 
the  present.     The  general  results  so  far  obtained  are  as  follows: 

Thermal  Death-Points, 
number  of  cultures  killed  at  various  temperatures. 


Temperature 
Cultures  .... 


50° 

55° 

60° 

65° 

70° 

75° 

2 

5 

24 

17 

16 

22 

80° 

I 


Pigment  formation. — The  production  of  color  by  the  bacteria 
is  not  only  markedly  affected  by  contemporaneous  conditions  of 
cultivation,  but  may  be  profoundly  modified  by  selective  action 
or  by  the  effect  of  antecedent  environment.  First,  of  the  conditions 
which  temporarily  affect  the  production  of  color,  without  modify- 
ing the  inherent  chromogenic  power  of  the  organisms,  may  be  men- 
tioned the  medium,  the  presence  of  free  oxygen,  and  the  tempera- 
ture. In  some  bacteria,  media  of  low  nutritive  value,  like  potato 
and  Nahrstoff,  appear  to  favor  pigment  formation,  but  with  the 
cocci  this  is  not  generally  the  case.  Agar  has,  on  the  whole,  shown 
a  better  development  of  chromogenesis  than  any  other  medium  tested. 
The  presence  of  free  oxygen  is  generally  an  essential  for  color  pro- 
duction, stab  growths  being  almost  invariably  lightly  colored.  We 
have  found  a  single  exception  to  this  rule  in  a  coccus  which  produces 


Generic  Characters  in  the  Coccaceae  1C9 

an  orange  pigment  of  much  deeper  tint  in  the  stab  than  on  the  sur- 
face. In  comparing  color  at  different  temperatures  we  have  found, 
in  general,  a  much  better  pigment  formation  at  20°  than  at  37°.  A 
deep  orange  growth  at  the  lower  temperature  may  often  correspond 
to  a  white  one  at  20°.  This  effect  has  been  recorded  in  our  routine 
studies,  and  will  be  more  fully  discussed  later  with  their  results  as  a 
basis.  Besides  these  temporary  modifications  of  the  chromogenic 
power,  the  actual  color  of  cultures  may  be  indirectly  affected  by 
certain  other  factors.  The  general  vigor  of  growth  is  naturally 
correlated  with  apparent  depth  of  color,  and  the  dryness  of  the  atmos- 
phere increases  its  intensity  by  evaporating  moisture  and  concen- 
trating the  pigment.  Both  these  factors,  increase  in  the  total  amount 
of  pigment  and  concentration  by  evaporation,  produce  a  pro- 
gressive deepening  of  color  in  old  cultures. 

Even  if  the  temporary  conditions  of  cultivation  are  quite  constant, 
the  chromogenic  power  of  an  organism  may  be  modified  by  its  pre- 
vious history.  In  thermal  death-point  observations  we  have  found 
interesting  cases  of  this  sort.  Some  streaks  made  from  broth  cul- 
tures which  had  been  exposed  to  a  temperature  of  50°  or  55°  were 
deeper  in  color  than  was  the  normal  for  the  organism,  but  in  most 
cases  they  were  much  lighter.  Sometimes  streaks  made  from  a  yel- 
low or  an  orange  chromogcn  after  such  treatment  were  almost  color- 
less, although  successive  transfers  generally  restored  the  normal 
properties.  Finally,  we  have  noticed  in  our  work  spontaneous 
variations  in  chromogenesis  such  as  have  been  recorded  by  Neumann 
(1897),  Conn  (1900),  and  Sullivan  (1905).  The  latter  authors  note 
that  on  a  plate  sown  from  a  single  colony  there  may  develop  colonies 
varying  appreciably  in  shade  from  which  selections  of  the  extremes 
will  produce  quite  distinct  types.  Neumann  records  the  sudden 
appearance  of  widely  different  strains,  as  sectors  in  old  and  carefully 
sealed  stab  cultures.  We  have  observed  both  phenomena  in  our 
cultures,  and  are  inclined  to  attribute  the  first,  and,  more  doubt- 
fully,  the   second,   to   variation   rather  than    contamination. 

In  spite  of  all  these  facts  it  is  clear  that,  as  the  cocci  normally 
occur  in  nature,  chromogenesis  is  one  of  their  most  distinct  and  sig- 
nificant differences.  In  any  series  of  plates  sown  with  washings 
from  the   skin  four   well-marked  types — red,    yellow,   orange,    and 


170  C.-E,  A.  WiNSLow  AND  Anne  F.  Rogers 

white — are  pretty  certain  to  occur.  We  have  therefore  included 
chromogenesis  as  one  of  our  routine  tests.  The  variations  due 
to  past  and  present  environment  are,  of  course,  easily  excluded  by 
the  maintenance  of  constant  conditions.  Our  stock  cultures  were 
in  all  cases  kept  on  agar  at  20°,  and  cultures  for  chromogenesis 
were  grown  on  that  medium,  and  at  that  temperature,  for  two  weeks. 
In  order  to  avoid  the  apparent  differences  due  to  the  vigor  of  growth 
or  to  evaporation,  a  portion  of  the  growth  was  removed  on  a  loop 
needle  and  spread  out  on  white  drawing-paper  with  a  rough  surface. 
After  drying  at  the  room  temperature,  the  color  was  compared  with 
an  arbitrary  standard  scheme. 

The  color  chart  used  for  matching  these  colors  was  devised  after 
a  very  careful  study  of  the  colors  actually  found  among  the  Cocca- 
ceae,  and  includes  nine  hues  ranging  from  white  through  lemon-yel- 
low, light  cadmium,  medium  cadmium,  lemon-yellow  and  cadmium 
orange,  red  and  cadmium  orange,  to  two  different  combinations 
of  red  with  lemon-yellow.  We  have  used  under  each  hue,  nine  differ- 
ent chromas,  obtained  by  successively  increasing  washes  of  the  hues 
on  white  paper.  The  hue  in  each  case  is  recorded  by  a  Roman 
numeral;    the  chroma,  or  number  of  wash,  by  an  Arabic  subscript. 

Liquefaction  0}  gelatin. — The  liquefaction  of  gelatin,  like  the  prop- 
erty of  pigment  production,  has  been  shown  to  be  subject  to  fluctu- 
ating variations.  Conn  (1900)  was  able  by  selection  to  obtain  from 
a  single  culture  of  a  milk  coccus  a  rapidly  liquefying  form,  and 
one  with  almost  no  peptonizing  power.  Smith  (1900)  records 
similar  experiences  with  colon  bacilli  and  forms  of  B.  proteus.  There 
appears  to  be  little  correlation  between  hquefaction  and  any  other 
power,  since  it  is  so  common  in  widely  separated  groups  of  bac- 
teria to  find  organisms  differing  in  this  respect,  while  identical  in 
all  other  properties. 

In  studying  liquefaction  we  have  determined  only  the  amount 
of  the  action  exerted  by  each  organism.  The  shape  of  the  liquefac- 
tion in  the  stab  culture  has  been  shown  by  Whipple  (1902)  to  vary 
within  the  widest  limits,  with  slight  differences  in  the  character 
of  the  medium,  and  the  Committee  on  Standard  Methods  (1905) 
has  omitted  this  character  from  its  list. 

For  determining  the  amount  of  liquefaction  we  have  used  the 


Generic  Characters  in  the  Coccaceae 


171 


method  suggested  by  Clark  and  Gage  (1905),  which  consists  in  inocu- 
lating gelatin  tubes  of  10  mm.  diameter  by  spreading  a  suspension 
of  the  culture  over  the  surface.  Liquefaction  proceeds  in  a  strati- 
form fashion,  and  its  amount  may  be  read  off  in  centimeters.  With 
such  a  method  one  may  determine  the  rapidity  of  hquefaction  either 


80 
70 
60 
50 
40 
50 
20 
10 


. 


0    .55       .85       1.55     1.85     2.55     2.85     5.55     585 

Fig.  4. — Liquefaction  of  gelatin  by  314  cocci.      Abscissae,  liquefaction  in  cm.      Ordinatcs,  number 
of  cultures. 

by  the  number  of  days  required  to  reach  a  fixed  point,  or  the  final 
amount  of  liquefaction.  In  general,  these  two  values  are  pretty 
closely  correlated,  but  in  a  preliminar)^  study  we  found  that  the 
final  differences  are  somewhat  sharper  as  well  as  easier  to  record. 
We  have  therefore  adopted  as  (jur  routine  measure  of  liquefying 
power  the  depth  of  liquefaction  after  30  days. 


172  C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 

Supplementary  tests. — Many  other  tests  than  those  mentioned 
are  sometimes  used  in  bacterial  diagnosis,  but  none  have  seemed 
suited  to  the  present  study.  The  questions  of  pathogenicity  and 
agglutinative  power  are  so  shrouded  in  confusion  as  to  be  unprom- 
ising. Meyer  (1902)  considered  serum  reactions  of  diagnostic 
value  among  the  streptococci,  and  Kolle  and  Otto  (1902),  and  Otto 
(1903),  obtained  good  results  with  the  staphylococci.  On  the  other 
hand,  Aronson  (1903),  Fischer  (1904),  and  Kerner  (1905),  after 
very  thorough  investigations,  came  to  the  conclusion  that  these  prop- 
erties among  the  streptococci  are  so  erratic  as  to  be  quite  valueless 
in  systematic  work.  From  a  general  survey  of  the  literature  of  the 
group,  it  seems  probable  that  the  properties  connected  with  infection 
and  immunity  are  hkely  to  be  too  easily  modified  to  prove  helpful 
in  classification. 

The  test  for  Mquefaction  of  starch  is  one  which  it  seems  logical 
to  include  with  those  which  show  the  relation  of  an  organism  to 
gelatin  and  the  sugars;  and  we  made  some  experiments  with  the 
starch  media  introduced  by  Smith  (1905).  It  appeared  that  cer- 
tain cocci  did  exert  an  amylolytic  action  and  the  study  of  this  char- 
acter would  probably  prove  of  considerable  interest.  It  has  been 
omitted  for  the  present,  for  lack  of  time. 

III.    RESULTS  OF  THE  INVESTIGATION. 

The  characters  observed  and  the  terms  in  which  they  are  record- 
ed may  be  summarized  as  follows : 

1.  Habitat. — Recorded  as  i  (diseased  conditions);  2  (normal 
body);  3  (water);  4  (earth);  or  5  (air).  The  significance  of  these 
various  habitats  has  been  more  fully  discussed  above.  It  should 
be  noted  that  Group  5  includes  certain  laboratory  cultures  whose 
origin  was  unknown. 

2.  Grouping  of  cells  and  dimensions. — Observed  in  stained  prepara- 
tions, made  from  20°  agar  cultures  less  than  five  days  old.  Group- 
ing recorded  as  i  (packets  present);  or  2  (packets  not  present). 
Extreme  dimensions  recorded  in  micromillimeters  to  the  nearest 
loth. 

3.  Relation  to  Gram  stain. — Observed  on  two  occasions  on  20° 
agar  cultures  less  than  five  days  old.     Treated  with  anihn-oil-gentian- 


Generic  Characters  in  the  Coccaceae  173 

violet  for  one  and  one-half  minutes;  Gram's  solution,  one  and  one- 
half  minutes;  95  per  cent  alcohol,  three  minutes.  Counterstained 
with  Bismarck  brown  for  one-half  minute.  Reaction  recorded 
as  —  (decolorized  in  both  tests);  ±:  (stained  once  and  decolorized  once); 
or  +  (stained  in  both  tests). 

4.  Vigor  oj  surface  growth  on  agar  streak  after  14  days  at  20°. — 
Recorded  as  i  (very  faint);  2  (meager);  3  (good);  4  (abundant); 
or  5   (very  heavy). 

5.  Amount  of  acid  produced  in  2  per  cent    dextrose  broth  after 

N 
14  days  at  20°. — Determined  by  titration  against  —  NaCl  in  the  cold 

with  phenolphthalein  as  an  indicator.  Recorded  value  is  the  difference 
between  inoculated  tubes  and  sterile  controls,  expressed  in  cubic 
centimeters  to  nearest  loth. 

6  .  Amount  of  acid  produced  in  2  per  cent  lactose  broth. — Same 
conditions  as  under  5. 

7.  Formation  of  nitrites  in  nitrate  solution. — Recorded  value 
is  the  number  of  tubes  giving  positive  test  for  nitrites  out  of  a  series 
of  10,  grown  for  seven  days  at  20°. 

8.  Formation  of  free  ammonia  in  nitrate  solution. — Same  method 
as  under  7. 

9.  Comparative  growth  after  14  days^  growth  on  agar  streak  at 
20°  and  57°,  respectively. — Recorded  as  i  (much  more  vigorous 
at  20°);  2  (more  vigorous  at  20°);  3  (equal);  4  (more  vigorous  at 
37°);  or  5  (much  more  vigorous  at  37°). 

10.  Chromo genesis. — Hue  and  chroma  of  pigment  produced  on 
agar  at  20°  after  14  days,  determined  by  comparison  with  color 
scheme  as  described  later. 

11.  Depth  in  cm.  of  gelatin  liquefaction  in  tube  of  i  mm.  diame- 
ter after  14  days  at  20°. 

It  would  be  well  to  extend  this  series  of  tests  by  a  study  of  the 
cell-grouping  in  broth,  motiUty,  fission  on  the  agar  block,  fermenta- 
tion of  saccharose,  effect  of  acid  and  alkaUne  media,  and  the  ther- 
mal death-point. 

I.  Habitat. 

The  distribution  of  the  cultures  isolated  among  the  various  habi- 
tats was  as  follows:   (i)  diseased  conditions,  59;  (2)  normal  body, 


174 


C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 


1 7° J  (3)  water,  95;  (4)  earth,  67;  (5)  air,  109.  It  is  probable  that 
this  deviates  from  a  representative  sampling  of  the  cocci  in  nature 
by  laying  too  great  stress  on  the  saprophytic  forms.  It  is  difficult 
to  find  cocci  at  all  in  earth  and  water,  whereas  they  are  present 
on  the  surfaces  of  the  body  in  enormous  numbers.  The  majority 
of  this  group  appear  to  be  parasitic  or  semiparasitic  in  habit.  At 
the  same  time,  the  fairly  equal  weight  given  to  the  saprophytic  forms 
helps  to  bring  out  the  differences  between  the  two  main  groups, 
those  living  in  or  on  the  animal  body  (i  and  2),  and  those  living  in 
the  outer  world  (3,  4,  and  many  of  5). 

We  have  prepared  tables  to  show  the  distribution  of  each  charac- 
ter among  various  habitats,  and  the  relations  shown  are  so  suggestive 
as  to  warrant  rather  full  discussion.  In  Table  i  is  given  the  cor- 
relation between  habitat  and  cell-grouping,  and  it  is  at  once  evident 
that  the  sarcinae  occur  in  greater  proportion  outside  than  inside  the 
body. 

In  this  and  succeeding  tables  the  figures  represent  the  number  of 
cultures  showing  each  combination  of  characters  out  of  the  500 
studied. 

TABLE  I. 

Correlation  between  Habitat  and  Cell-Grouping. 


Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

No.  packets 

Packets 

44 
15 

145 

25 

45 
50 

33 
34 

78 
31 

Whereas  packets  are  more  abundant  in  earth  and  water,  the 
other  forms — chains,  plates,  and  irregular  groups  without  sarcinae 
— make  up  two-thirds  or  more  of  the  parasitic  forms. 

TABLE  2. 
Correlation  between  Habitat  and  Gram  Stain. 


Gram  Stain 

Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

_ 

14 
17 

28 

12 

50 
108 

46 
29 
20 

37 
18 
12 

36 

± 

+   

32 
41 

The  distribution  of  our  cultures  according  to  their  relation  to 
the  Gram  stain  brings  out  a  similar  condition.     The  cultures  giving 


Generic  Characters  in  the  Coccaceae 


/3 


a  consistent  positive  reaction  make  up  far  more  than  half  the  total 
among  the  parasitic  forms  from  the  first  two  habitats,  and  less  than 
one-fourth  of  the  forms  from  water  and  earth.  The  air  cultures 
in  almost  all  cases  exhibit  an  intermediate  relation,  as  would  be  ex- 
pected, since  they  must  contain  forms  from  both  sources.  A  positive 
reaction  to  the  Gram  stain  is  evidently  closely  correlated  with  life 
in  and  on  the  animal  body. 

TABLE  3. 

Correlation  between  Habitat  and  Surface  Growth. 


Surface  Growth 

Very  faint 

Meager 

Good 

Abundant 

Very  heavy 


Diseased 
Conditions 


o 
I 

27 
8 


Normal  Body 

Water 

Earth 

Air 

16 

3 

0 

0 

IS 

2 

3 

I 

98 

38 

24 

33 

34 

38 

3J 

60 

7 

14 

10 

16 

The  abundance  of  surface  growth  also  varies  with  the  habitat. 
Very  faint  and  meager  growths  are  fairly  abundant  in  the  forms 
from  the  surfaces  of  the  body,  as  would  be  expected,  since  our  culture 
media  are  unfavorable  for  the  more  strictly  parasitic  forms.  On 
the  other  hand,  a  majority  of  the  earth  and  water  cocci  show  abun- 
dant or  very  heavy  surface  growths.  The  air  forms  are  character- 
ized by  particularly  abundant  development,  as  would  naturally  be 
expected,  since  only  the  vigorous  cells  probably  survive  drj-ing  and 
dispersal  through  the  air. 

TABLE  4. 
Correlation  between  Habitat  and  the  Fermentation  of  Dextrose  Broth. 


Acid   Production   per   Cent    of 
Normal 

Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

0 . 0  and  alkaline 

12 

7 
20 
17 

3 

12 

33 

82 

38 

S 

39 
33 
19 

13 
3 

28 
24 
10 

S 
0 

20 
34 

0  3—0 .6     

25 

0.  7-2 .0 

2 . 0  and  over 

34 
6 

TABLE  s- 
Correlation  between  Habitat  and  the  Fermentation  of  Lactose  Broth. 


Acid  Production    per  Cent  of 
Normal 

-0.3  and  more  alkaline 

-o .  1  and  0.0 

0.1-0.4  

o.S-i-4  

1 . 5  and  over 


Diseased 
Conditions 


6 

23 

IS 

II 

4 


Normal  Body 


13 

37 

64 

40 

8 


Water 


Q 

63 
16 

5 
3 


Earth 


10 
43 
II 

4 
o 


Mr 


13 
40 
32 
19 
5 


176 


C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 


In  examining  Tables  4  and  5,  which  show  the  fermentative  power 
of  dextrose  and  lactose  broth,  the  fundamental  difference  between  the 
parasitic  and  saprophytic  cocci  is  again  made  evident.  In  the  first 
two  groups,  from  the  animal  body,  over  two-thirds  of  the  cultures 
produce  more  than  0.3  per  cent  of  normal  acid;  while  among  the 
earth  and  water  forms  two-thirds  of  the  organisms  form  less  than  this 
amount.  With  lactose  the  same  law  holds.  Two-thirds  of  the  cocci 
from  the  normal  body  produce  acid  in  lactose,  against  less  than 
one-third  of  the  water  and  earth  forms.  The  air  cultures  show  an 
intermediate  relation. 

TABLE  6. 
Correlation  between  Habitat  and  Reduction  of  Nitrates. 


Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

44 
10 

7 

147 
7 

75 
8 

44 
10 

13 

72 
18 
30 

Nitrites  formed 

Ammonia  formed        .        ... 

The  property  of  nitrate  reduction  does  not  appear  to  be  related 
to  habitat  in  any  such  direct  way  as  the  other  characters  studied. 
The  air  cocci,  however,  show  a  peculiarity  of  considerable  interest, 
nitrite  formation  being  common,  and  ammonia  formation  very  com- 
mon, among  them. 

TABLE  7. 
Correlation  between  Habitat  and  Optimum  Temperature  for  Growth. 


Optimum 

20" 

20°  or  37° 

37°   


Diseased 
Conditions 


9 
36 
14 


Normal  Body 


II 
112 

47 


Water 


29 
10 


Earth 


23 

42 

2 


Air 


II 

89 

9 


While  a  majority  of  the  cultures  studied  grow  indifferently  at  20° 
or  37°,  it  appears  from  Table  7  that  among  the  parasitic  forms  a 
fair  proportion  are  favored  by  the  body  temperature,  while  more 
of  the  earth  and  water  forms  develop  best  at  20°.  With  regard  to 
the  optimum  temperature  for  color  formation,  no  definite  relation 
with  habitat  appears,  except  as  involved  in  the  double  relation  between 
chromogenesis  and  habitat,  and  chromogenesis  and  the  optimum 
temperature  for  color  formation.  These  figures  are  therefore 
omitted. 


Generic  Characters  in  the  Coccaceae 

TABLE  8. 
Correlation  between  Habitat  and  Chromocenisir. 


177 


Chromogenesis 

Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

White 

4 
33 
21 

I 

«7 

37 

116 

0 

S 

76 

6 

8 

I 

10 
6 

13 
S8 
aS 

Yellow 

Orange 

Red 

10 

Table  8  brings  out  some  of  the  most  definite  relations  yet  con- 
sidered, between  habitat  and  chromogenesis.  It  is  evident  that  the 
white  and  orange  forms  are  largely  parasitic,  and  the  yellow  and  red 
forms  as  distinctively  saprophytic.  More  than  half  of  the  white 
and  more  than  two-thirds  of  the  orange  chromogens  come  from  the 
first  two  habitats,  while  only  one-third  of  the  yellow  forms  have  such 
an  origin.  The  distribution  of  the  red  pigment-formers  is  even  more 
notably  saprophytic.  Only  one  culture  out  of  25  occurred  among 
the  229  cultures  from  the  body. 

TABLE  9. 
Correlation  between  Habitat  and  Gelatin  Liquefaction. 


Gelatin  Liquefaction  (Depth  in 
cm.) 

Diseased 
Conditions 

Normal  Body 

Water 

Earth 

Air 

0  0 

13 
24 
22 

68 
36 
66 

43 
42 
10 

26 
30 
II 

36 
45 

28 

0.  i-i .5 

1 . 6  and  over 

The  table  for  habitat  and  gelatin  liquefaction  (Table  9)  shows 
this  property  occurring  among  the  earth  and  water  forms  to  a  less 
degree  than  among  the  parasitic  cocci.  This  fact,  and  the  fact 
that  the  parasitic  forms  are  high  acid-producers,  as  noted  above, 
are  of  practical  significance  in  connection  with  the  bacteriological 
analysis  of  water.  It  has  long  been  suspected  that  acid  producti(m 
and  gelatin  liquefaction  were  associated  with  intestinal  organisms, 
and  we  have  here  a  measure  of  the  truth  of  this  proposition,  in  the 
case  of  the  cocci  at  least.  From  the  food  conditions  which  obtain 
in  the  ahmentary  tract,  and  to  a  less  extent  on  the  outer  surfaces 
of  the  body,  it  is  natural  that  these  properties  should  be  highly 
developed. 

The  forms  from  Habitat  II  (the  surfaces  of  the  normal  body) 
group  themselves  most  abundantly  at  two  extremes,  68  being  non- 


178  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

liqueficrs  and  66  active  liquefiers,  while  only  36  occupy  the  inter- 
mediate position,  which  is  of  most  frequent  occurrence  in  all  the  other 
habitats.  It  is  probable  that  this  may  be  accounted  for  by  the  pres- 
ence, in  Habitat  II,  of  two  distinct  series — the  white  and  colorless 
forms,  which,  as  we  shall  see  later,  are  non-liquefiers,  and  the  orange 
forms,  which  peptonize  strongly. 

From  a  general  survey  of  our  habitat  studies  it  is  evident  that 
the  forms  from  the  body  exhibit  quite  different  characteristics  from 
those  of  the  water  and  earth  cocci.  The  parasitic  forms  generally 
react  positively  to  the  Gram  stain,  give  only  fair  surface  growths 
on  the  surface  of  artificial  media,  produce  acid  in  dextrose  and  lac- 
tose, grow  best  at  37°,  produce  no  pigment  or  a  white  or  an  orange 
pigment,  and  Hquefy  gelatin.  The  saprophytes,  on  the  other  hand, 
are  more  apt  to  occur  in  packets,  to  be  Gram-negative,  to  grow 
abundantly  on  artificial  media,  best  at  20°,  to  produce  yellow  and 
red  pigments,  and  to  exert  little  action  on  sugars  and  gelatin.  The 
air  cocci  are  generally  intermediate  in  character  between  the  two 
groups,  but  show  special  powers  of  nitrate  reduction. 

2.  Grouping  of  Cells,  and  Dimensions. 

The  cocci,  as  noted  above,  were  divided  into  two  classes  only, 
according  to  the  character  of  the  cell  aggregates;  155  cultures  showed 
the  packets  or  sarcina-grouping,  and  345  did  not. 

TABLE  10. 
Correlation  between  Cell-Grouping  and  Gram  Stain. 


Gram  Stain 

Irregular  Groups 
and  Chains 

Packets 

75 
98 

172 

± 

+ 

48 

37 

We  have  pointed  out  above  that  packets  are  most  abundant  among 
the  saprophytic  cocci  of  the  earth  and  water.  Table  10  shows 
the  relation  between  cell-grouping  and  the  Gram  stain,  clearly 
indicating  that  the  packets  tend  to  be  Gram-negative,  while  a  ma- 
jority of  the  other  forms  give  a  positive  reaction. 

Table  11  shows  a  distinct  correlation  between  cell-grouping  and 
the  vigor  of  surface  growth.     A  large  majority  of  the  non-packet- 


Generic  Characters  in  the  Coccaceae 


179 


forming  organisms  produce  only  very  faint,  meager,  or  good  growths 
while  a  large  majority  of  the  sarcinae  form  abundant  or  very  heavy 

TABLE  II. 
Correlation  between  Cell-Grouping  and  Surface  Growth. 


Surface  Growth 

Irregular  Groups 
and  Chains 

Packets 

18 

16 

169 

132 

10 

I 

Meager 

Good 

Abundant 

5 
46 
58 

45 

growths.     The  fact  that  the  packet-forms  flourish  on  artificial  media 
should  naturally  result  from  their  saprophytic  origin. 

TABLE  12. 
Correlation  between  Cell-Grouping  and  the  Fermentation  of  Dextrose  Broth. 


Add  Production 
(Per  Cent  Normal) 

Irregular  Groups 
and  Chains 

Packets 

0  and  under 

0.1-0.2 

0.3-o.e 

58 
S6 
128 
87 
16 

53 
54 
27 

2 . 0  and  over 

I 

TABLE  13. 
Correlation  between  Cell-Groupino  and  the  Fermentation  of  Lactose  Broth. 


Acid  Produrtion 
(Per  Cent  Normal) 

Irregular  Groups 
and  Chains 

Packets 

—  0.2  and  more  alkaline 

0. 1  and  0.0 

0.1-0.4  

0.5-1.4  

I .  s  and  over 

38 

"3 

X04 

72 

18 

II 

01 

35 
16 

2 

A  marked  correlation  between  the  packet-forming  organisms 
(presumably  saprophytic)  and  the  fermentation  of  sugars  is  mani- 
fested in  Tables  12  and  13.  Taking  Table  12  as  an  illustration, 
there  will  be  found  to  be  about  60  per  cent  of  the  packet-formers 
producing  alkali,  or,  at  most,  fermenting  dextrose  but  slightly  (to 
0.2) — almost  none  of  the  organisms  occurring  in  the  class  of  highest 
acid  producers.  Conversely,  70  per  cent  of  the  organisms  which 
do  not  form  packets  produce  acid  from  0.3  up  to  the  highest  amount. 

The  power  to  reduce  nitrates  appears  with  about  equal  regular- 
ity in  both  our  morphological  groups. 


i8o 


C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 


TABLE  14. 
Correlation  between  Cell-Grouping  and  Optimum  Temperature  for  Growth. 


Optimum  Temperature 

20° 

20°  or  37° 

37° 


Irregular  Groups 
and  Chains 


40 

237 
68 


Packets 


43 
98 
14 


A  slight  but  distinct  correlation  appears  between  grouping  and 
optimum  temperature  for  growth.  In  each  case  most  of  the  cultures 
grow  at  20°  or  37°  indifferently;  but  a  fair  proportion  of  the  more 
saprophytic  sarcinae  show  better  development  at  20°,  while  among 
the  other  forms  more  find  an  optimum  at  37°  than  at  20°.  With 
regard  to  the  optimum  temperature  for  color  formation  there  is  a 
slight  difference,  only  two-fifths  of  the  sarcinse  showing  more  chromo- 
genesis  at  20°  than  at  37°,  against  one- half  of  the  other  cultures. 
This,  as  we  shall  see  later,  is  probably  connected  with  a  difference 
in  chromogenesis. 

TABLE  IS. 
Correlation  between  Cell-Grouping  and  Gelatin  Liquefaction. 


Gelatin  Liquefaction 

(cm.) 

Irregular  Groups 
and  Chains 

Packets 

00....            

127 
no 
108 

59 
67 
29 

0. 1— 1 . 5  cm 

Table  15,  for  the  relation  of  gelatin  liquefaction  to  morphology, 
shows  only  that  the  highest  grades  of  liquefaction  are  somewhat 
less  numerous  among  the  packets  than  in  the  other  group. 

The  results  obtained  with  regard  to  the  size  of  the  individual 
cell  were  much  less  suggestive  than  the  facts  concerning  cell-grouping. 
Dimensions  were  measured  in  all  cases  on  the  stained  specimens, 
and  were  recorded  independently  on  at  least  two  occasions.  The 
attempt  was  made  to  note  in  each  case  the  extreme  diameters  observed, 
and  the  values  finally  adopted  represent  the  average  between  the 
recorded  extremes.  Individual  cells  ranged  from  o.i  to  2 . o a*-. 
With  the  packets  it  was  found  impossible  to  determine  the  maximum 
size  of  the  single  cell,  on  account  of  the  frequent  occurrence  of  small, 
recently  formed  packets  which  stained  as  a  whole  like  one  cell. 
We  never  felt  certain  that  what  appeared  like  a  large  cell  was  not 


Generic  Characters  in  the  Coccaceae 


i8i 


really  a  group  of  eight  small  ones.  The  sarcinai  in  general  showed 
quite  small  individual  units,  o.  i  or  0.2  /*  as  a  rule,  with  no  constant 
deviations.  The  packets  are  therefore  entirely  omitted  from  the 
consideration  of  dimensions. 

The  average  sizes  of  the  345  cultures,  not  occurring  in  packets, 
are  grouped  in  convenient  classes  in  Table  16  and  plotted  in  Fig.  i. 

TABLE  16. 
Dimensions  of  345  Cocci. 


Size,  average,  w  .  .  .  . 
Number  of  cultures 


0. 1 

0.  2 

03 

0.4 

OS 

0.6 

0.7 

0.8 

0.0 

22 

82 

"3 

60 

40 

12 

7 

6 

I 

1 .0 

2 


The  sizes  of  the  cocci  studied  are  evidently  distributed  on  a  fairly 
normal  curve  of  frequency.  The  mode  is  at  0.3^  and  the  curve 
is  markedly  skew,  with  infinite  extension  toward  the  larger  sizes. 
The  important  practical  point  is  that  the  forms  measured  appear 
to  behave  like  a  fairly  homogeneous  series  var)'ing  about  a  single 
mode. 

We  have  made  tentative  calculations  of  correlation  between  cell 
dimensions  and  other  characters,  with  almost  entirely  negative  result. 
The  only  property  showing  any  relation  is  that  of  gelatin  liquefaction. 

TABLE  17. 

CORREI.ATION    BETWEEN    CeI.L    DIMENSIONS    AND    CeLATIN    LIQUEFACTION. 


Gelatin  Liquefaction 
(cm.) 

Maximum  Size 
0.  3/i  and  under 

Maximum  Size 
over  0 .  3  M 

47 
6s 
69 

78 

0.1-1.5  

47 
^9 

An  appreciable  inverse  correlation  is  shown  between  the  size 
of  the  cell  and  the  rate  of  gelatin  liquefaction,  the  smaller  cocci 
liquefying  most  readily. 

3.  Gram  Stain. 

We  have  pointed  out  above  that  the  reaction  of  the  cocci  to  the 
anilin-oil-iodin  stain  is  a  variable  character,  many  forms  showing  a 
positive  reaction  on  one  occasion  and  a  negative  reaction  when  next 
tested.  Nevertheless  the  test,  variable  as  it  is,  shows  quite  constant 
relations  to  other  characteristics;  and  we  feel  convinced  that  among 
the  cocci  where  all  characters  are  more  or  less  fluctuating  any  prop- 


l82 


C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 


erty  which  on  the  average  shows  a  definite  correlation  with  other 
properties  has  systematic  significance. 

Of  the  cocci  studied,  145  showed  in  two  tests  a  Gram  negative 
reaction  on  both  occasions,  and  209  two  positive  reactions,  while 
146  were  once  stained  and  once  decolorized.  Grouped  thus  in 
three  divisions,  we  have  seen  that  the  positive  reaction  is  charac- 
teristic of  the  parasitic  forms,  while  saprophytic  forms  and  packets 
tend  to  be  Gram  negative.  With  surface  growth  only  an  insignifi- 
cant relation  appears,  the  less  richly  growing  forms  showing  a  slightly 
higher  proportion  of  positive  reactions. 

TABLE  18. 
Correlation  between  Gram  Stain  and  the  Fermentation  of  Dextrose  Broth. 


Acidity  Produced 
(Per  Cent  Normal) 

o .  o  and  over 

o. i-o. 2  

0.3-0.6  

o. 7-2.0  

2 . 1  and  over 


Gram  Negative 

Gram  Variable 

Gram  Positive 

49 

44 

18 

48 

29 

33 

35 

4S 

75 

12 

26 

69 

I 

2 

14 

TABLE  19. 
Correlation  between  Gram  Stain  and  the  Fermentation  of  Lactose  Broth. 


Acidity  Produced 
(Per  Cent  Normal) 

Gram  Negative 

Gram  Variable 

Gram  Positive 

-0.2  and  more  alkaline 

-0 . 1  and  0.0 

0.  i-o. 4 

0.5-1-4  

1 . 5  and  over 

20 
72 
34 
19 
0 

14 
82 
24 
20 
6 

IS 
50 
81 
49 
14 

The  relation  between  the  Gram  reaction  and  the  fermentation 
of  carbohydrates  is  a  surprisingly  close  one.  Each  line  in  Tables 
18  and  19  showing  the  distribution  of  organisms  among  the  grades 
of  acidity  forms  a  regular  curve.  In  each  case  the  mode  of  the 
Gram-negative  cultures  occurs  at  the  neutral  point,  and  that  of  the 
Gram- positive  cultures  at  a  moderately  high  acidity,  with  the  doubt- 
ful cultures  showing  an  intermediate  relation. 

TABLE  20. 
Correlation  between  Gram  Stain  and  Optimum  Temperature  for  Growth. 


Optimum  Temperature 

Gram  Negative 

Gram  Variable 

Gram  Positive 

20° 

34 
103 

8 

29 
89 
28 

20 

20°  or  ^7°  

143 

1170 

46 

Generic  Characters  in  the  Coccaceae 


183 


With  nitrate  reduction  and  the  optimum  temperature  for  chro- 
mogenesis  the  Gram  reaction  shows  no  special  relations.  With 
optimum  growth  temperature,  on  the  other  hand,  Table,  20  shows 
a  distinct  connection.  As  always,  most  of  the  cuUures  grow  equally 
at  both  temperatures.  Among  the  decolorized  cultures,  however, 
a  fair  proportion  grow  best  at  20°,  while  with  the  positive  forms 
37°  is  most  favorable.  Such  a  relation  would,  of  course,  be  expected 
from  the  generally  saprophytic  habit  of  the  negative  forms. 

The  liquefaction  of  gelatin  does  not  show  any  distinct  relation 
to  the  Gram  reaction.  On  the  whole,  therefore,  we  may  conclude 
that  the  cocci  which  decolorize  by  Gram  are  generally  earth  and 
water  forms,  which  notably  fail  to  ferment  sugars,  and  which  grow 
best  at  20°.  The  marked  correlation  with  the  power  of  acid  pro- 
duction, in  the  absence  of  other  equally  marked  relations,  seems 
to  invite  further  study  of  the  physiological  basis  of  these  properties. 


4.  Surface  Growth. 

The  cocci  studied  were  divided  into  five  groups  according  to  the 
vigor  of  surface  growth  on  agar.  The  first  group,  of  "very  faint" 
growths,  includes  19;  the  second  group,  of  "meager"  growths, 
includes  21  forms;  "good"  and  "abundant"  growths  occur  215 
and  190  times,  respectively;  and  55  cocci  show  "very  heaNy" 
growths. 

TABLE  21. 
Correlation  between  Surface  Growth  and  the  Fermentation  of  Dextrose  Broth. 


Acidity  Produced  (Per  Cent 
Normal) 


o  and  alkaline 

I-0.2    

3-06   

7-2 .0  

o  and  over  . . . 


Very  Faint 


Meager 


2 

3 
II 

4 
I 


Good 


42 
4> 
76 

S3 
3 


Abundant 


41 
51 
54 
39 
5 


Very  Heavy 


33 

14 
12 

s 

I 


TABLE  22. 
Correlation  between  Surface  Growth  and  the  Fermentation  of  Lactose  Broth. 


Acidity  Produced  (Per  Cent 
Normal) 

-0.2  and  more  alkaline 

— o .  I  and  0.0 

o. 1-0.4 

0.5-1.4 

1 . 5  and  over 


Very  Faint 

Meager 

Good 

Abund 

2 

3 

18 

22 

2 

5 

80 

87 

s 

^ 

6s 

49 

2 

4 

47 

a? 

8 

I 

5 

S 

Abundant       \'ery  Heav-y 


5 

30 

It 

8 

I 


1 84 


C.-E.  A,  WiNSLOw  AND  Anne  F.  Rogers 


We  have  seen  above  that  the  fainter  growths  are  characteristic 
of  the  parasitic  forms,  and  the  heavier  ones  of  the  earth  and  water 
cocci.  The  heavier  growths  are  more  common  among  the  packets 
than  with  other  cell  groupings. 

In  comparing  the  vigor  of  surface  growth  with  the  fermentation 
of  carbohydrates,  a  distinct  relation  appears  at  the  ends  of  the  scale, 
although  the  bulk  of  the  growth,  under  the  headings  "good"  and 
"abundant,"  exhibit  uniform  characteristics.  The  "very  faint" 
growths,  which  denote  members  of  the  genus  Streptococcus,  as  pre- 
viously defined,  are  associated  with  a  maximum  of  acid  production 
falling  in  the  highest  acidity  class  in  each  sugar  table.  On  the  other 
hand,  the  "very  heavy"  growths  are  mainly  forms  which  fail  to  act 
on  either  sugar. 

TABLE  23. 
Correlation  between  Surface  Growth  and  Nitrate  Reduction. 


Very  Faint 

Meager 

Good 

Abundant 

Very  Heavy 

No  reduction 

19 
0 
0 

18 
2 

I 

173 
25 
23 

133 
38 
29 

39 

Nitrites  formed 

5 

Ammoniti  formed 

12 

Surface  growth  and  nitrate  reduction  show  a  suggestive  relation. 
Among  the  very  faint  growths  of  the  Streptococcus  type  no  reduc- 
tion occurs,  and  almost  none  among  the  "meager"  forms.  The 
"good"  and  "abundant"  groups  show  an  increasing  proportion 
of  reducing  organisms,  and  the  "very  heavy"  group  shows  many 
ammonia-formers  and  a  fair  proportion  of  nitrite  production. 

TABLE  24- 
Correlation  between  Surface  Growth  and  Gelatin  Liquefaction. 


Gelatin  Liquefaction 
(Depth  in  cm.) 

Very  Faint 

Meager 

Good 

Abundant 

Very  Heavy 

0.0 

19 
0 
0 

14 
6 

I 

63 
84 
68 

80 
62 
48 

10 

o.i-i.s 

1 . 6  and  over 

25 

20 

With  gelatin  liquefaction  there  exists  the  same  group  correlation 
manifest  for  the  other  characters.  The  "very  faint"  group  shows 
not  one  liquefier,  and  the  "meager"  group  very  few,  while  the  more 
vigorous  forms  exhibit  a  more  even  distribution. 

In  general,  our  study  of  surface  growth  brings  out  two  distinct 


Generic  Characters  in  the  Coccaceae  185 

groups  of  organisms.  The  first  group,  including  the  forms  with 
faint  or  meager  surface  development,  corresponds  to  the  genus 
Streptococcus  as  defined  above.  It  is  sharply  characterized  by  high 
acid  production  and  the  absence  of  gelatin  liquefaction  or  nitrate 
reduction.  Cocci  of  this  type  are  characteristically  parasitic,  and 
very  rarely  show  the  sarcina  grouping.  On  the  other  hand,  the 
more  vigorous  forms  are  generally  saprophytic,  and  frequently 
show  packets.  They  ferment  sugar  slightly  or  not  at  all,  and  often 
reduce  nitrates  and  liquefy  gelatin. 

5.   Fermentation  of  Carbohydrates. 

In  measuring  the  amount  of  acid  produced  in  dextrose  and  lactose 
broth,  two  check  determinations  were  made  in  each  case,  and  the 
figure  finally  recorded  was  the  average  of  these  two  determinations. 
The  correspondence  between  the  two  tubes  was  generally  close. 
From  150  cases  for  each  sugar  we  have  calculated  the  probable  error 
of  a  single  observation,  and  find  it  only  ±0.043  for  dextrose  and 
±0.036  for  lactose.  Since  our  readings  were  only  taken  to  loths 
of  a  c.c,  it  is  evident  that  even  a  single  determination  would  be 
sufficiently  accurate  for  any  long  series. 

The  general  results  of  the  titrations  are  shown  in  Table  25  and 
in  Fig.  2.  It  will  be  noticed  that  with  both  acids  the  organisms  are 
ranged  with  fair  regularity  about  a  single  mode.  The  majority 
of  the  cultures  studied  produce  an  acidity  of  0.1-0.2  per  cent 
normal  in  dextrose,  and  fail  to  ferment  lactose  at  all.  In  cither 
case  a  few  cultures  only  show  an  alkaline  reaction,  and  with  lactose 
less  than  half  of  the  organisms  form  acid,  giving  the  curve  for  that 
sugar  a  very  acute  form.  The  curve  for  dextrose  falls  off  much 
more  slowly,  and  shows  slight  secondary  modes  at  acidities  of 
0.5-0.6  per  cent  normal  and  1.3-1,4  per  cent  normal.  Finally, 
both  curves  show  an  extraordinary'  extension  in  the  direction  of 
the  higher  acidities.  It  will  be  noticed  that  for  each  sugar  several 
of  the  highest  reactions,  ranging  from  3  to  nearly  10  per  cent  normal, 
have  been  omitted  from  the  chart.  We  may  fairly  consider  the 
fermentation  of  the  two  carbohydrates  together,  since,  as  shown 
in  Table  26,  they  are  very  closely  correlated.  The  amount  of  acid 
produced  in  lactose  broth  is  almost  always  less  than  that  in  dextrose 
broth,  but  the  two  vary  together. 


[86 


C.-E,  A.  WiNSLow  AND  Anne  F.  Rogers 


TABLE  25. 
Acid  Production  in  Sugar  Broths. 


Acidity 
Produced 

Number  of  Cultures  in  Each  Group 

(Per  Cent 

Normal) 

— O.0-O.8 

—  0.7-0.6 

-0.5-0.4 

—  0.3-0.2 

—  0.  i-o.o 

0. 1-0.2 

0.3-0.4 

0.5-0.6 

Lactose 

Dextrose 

I 

I 

7 
4 

41 
9 

204 

98 

86 
no 

52 
80 

44 
76 

Lactose  . 
Dextrose 


o . 7-0 . 


23 
45 


o.g-i .0 


10 
28 


7 
12 


13-1-4 


4 
13 


1.5-1.6 


I. 7-1 -8 


1 . 9-2 . o 


2.3-2.4 


2.5-2.6 

2.7-2.8 

2 . 9-3 • 0 

3 • 1-3  •  2 

3  -3 

4.0 

4-3 

4.6 

4-7 

S-9 

8.2 

8.6 

9   7 

Lactose 

I 

3 

I 
2 

0 

2 

I 

I 

2 

I 

I 

I 

I 

I 

Dextrose 

I 

TABLE  26. 
Correlation  between  Fermentation  of  Dextrose  and  Lactose  Broths. 


Dextrose  Acid  Production 
(Per  Cent  Normal) 

Lactose 

—  0.2  and  more 

alkaline 

Lactose 
—  0.1  and  0 

Lactose 
0. i-o. 4 

Lactose 
0.5-1.4 

Lactose 
1 . 5  and  over 

0  and  alkaline 

0 . 1  and  0.2 

0.3-0.6  

0 . 7—2 .0  

19 

II 

II 

9 

0 

72 
57 
47 
26 
2 

15 
29 
55 
36 
I 

4 
13 
40 
29 

2 

I 
0 
I 
6 

2.0  and  over 

12 

We  have  noted  before  that  the  power  of  carbohycirate  fermenta- 
tion is  specially  characteristic  of  the  parasitic  cocci,  and  of  those 
which  do  not  show  the  packet  grouping.  The  high  acidities  are 
also  correlated  with  a  positive  reaction  to  the  Gram  stain  and  with 
a  faint  surface  growth  on  artificial  media. 

TABLE  27. 
Correlation  between  Fermentation  op  Dextrose  Broth  and  Nitrate  Reduction. 


Dextrose  Acid  Pro- 
duction (Per  Cent 

Normal) 

No  Reduction 

Nitrite  Found 

Ammonia  Found 

0  and  alkaline 

0. I  and  0.2  

0.3-0. 6 

75 
86 

115 
90 
16 

13 
II 
28 
18 
0 

16 
16 
18 

0.7-2.0 

2 . 0  and  over 

15 
0 

Table   27,  for  the  relation  between  nitrate  reduction  and  acid 
formation  in  dextrose  broth,  shows  only  that  the  class  of  strong 


Generic  Characters  in  the  Coccaceae 


187 


acid-formers  fail  entirely  to  form  nitrites  and  ammonia.  A  correla- 
tion table  for  lactose,  which  we  have  not  thought  it  necessar)'  to  quote 
here,  shows  a  similar  relation.  The  high  acid-formers,  it  may  be 
remembered,  belong  to  the  genus  Streptococcus^  with  its  weak  power 
of  growth  on  artificial  media. 

Our  tables  of  the  correlation  between  carbohydrate  fermentation 
and  optimum  temperature  fail  to  show  any  striking  coincidences. 
There  is  an  appreciable  tendency  for  the  higher  acid-formers  to 
grow  better  at  37°,  and  for  the  alkaline  or  neutral  forms  to  grow  bet- 
ter at  20° ;  but  we  have  not  considered  this  important  enough  to  war- 
rant the  reproduction  of  the  tables. 

TABLE  28. 
Correlation  between  Gelatin  Liquefaction  and  Fermentation  of  Dextrose  Broth. 


Acid  Production 
(Per  Cent  Normal) 

Gelatin 
Not  Liquefied 

Gelatin   Liquefied 
(0.1-1.5  cm.) 

Gelatin  Liquefied 
(1.6  cm.  and  over) 

0  and  alkaline 

o.iando.2 

0.3-0.6 

0. 7-2.0 

2 .0  and  over 

38 
45 
47 
43 
13 

61 
42 
37 
36 

I 

12 
23 
72 
27 
3 

TABLE  29. 
Correlation  between  Gelatin  Liquefaction  and  Fermentation  of  Lactose  Broth. 


Acid  Production 
(Per  Cent  Normal) 

Gelatin  Not 
Liquefied 

Gelatin   Liquefied  Gelatin  Liouefied 

(0.1-1.4  cm.)      (1.5  cm.  and  over) 

1 

-0.2  and  more  alkaline 

-0 . 1  and  0 

0. 1-0.4 

0.5-1  4 

1 . 5  and  over 

20 
91 
49 
13 
13 

19 
86 
46 
25 

I 

n 
27 
43 
SO 
6 

The  relation  between  the  organisms  which  ferment  the  sugar  broths 
and  liquefy  gelatin  is  shown  in  Tables  28  and  29.  These  tables 
may  be  considered  together,  as  they  reveal  practically  the  same  law. 
The  relation  between  acid  production  and  gelatin  liquefaction  is 
evidently  a  somewhat  complex  one.  The  forms  which  fail  to  ferment 
carbohydrates  for  the  most  part  exhibit  a  moderate  amount  of  lique- 
faction. Next  comes  a  group  of  the  moderate  acid-producers  which 
liquefy  most  actively.  Finally,  the  highest  acid-formers  are  mostly 
non-liqueficrs.  We  shall  get  more  light  on  these  three  groups  when 
we  come  later  to  consider  the  classes  of  the  cocci  according  to  their 
chromogenesis. 


1 88 


C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 


To  our  conception  of  the  non-acid-forming  cocci  as  typically 
saprophytic  organisms  frequently  occurring  in  packets  and  usually 
Gram-negative,  we  may  add  the  property  of  moderate,  but  not 
very  active,  liquefaction  of  gelatin.  The  very  high  acid-producers 
are  generally  parasitic,  do  not  show  sarcinae,  stain  by  Gram,  grow 
faintly  on  agar,  and  fail  to  reduce  nitrates  or  liquefy  gelatin.  Between 
these  two  groups  is  a  third  type  which  forms  a  moderate  amount 
of  acid  and  produces  the  most  active  liquefaction  of  gelatin. 

6.  Reduction  of  Nitrates. 

As  described  above,  the  tests  for  nitrate  reduction  were  made  in 
parallel  in  lo  tubes,  and  a  marked  variation  was  found  in  the  indi- 
vidual tubes,  as  shown  in  Table  30.  This  is  perhaps  to  be  expected, 
since  the  development  of  bacteria  in  such  a  nutrient  medium  as  nitrate 
solution  must  be  subject  to  many  chance  variations  in  the  number 
and  vigor  of  the  organisms  inoculated. 


TABLE  30. 
Reduction  of  Nitrates. 


Number  of  Tubes  Showing 
Positive  Tests   .          

I 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Nitrites 

Ammonia 

27 
30 

14 
26 

8 
21 

5 
15 

II 
16 

5 
10 

II 
2 

7 
11 

12 
4 

24 
22 

Table  30  shows  a  considerable  number  of  cultures  yielding  posi- 
tive results  in  one  or  two  out  of  the  10  tubes  tested,  less  in  from  four 
to  seven  of  the  tubes,  and  more  again  giving  check  results  in  all 
10  tubes.  In  order  to  compare  this  property  with  others,  it  was 
necessary  to  distinguish  between  positive  and  negative  cultures,  and 
we  have  therefore  considered  the  test  to  be  positive  when  five  or 
more  of  the  tubes  showed  some  reduction.  The  cultures  then  grouped 
themselves  into  three  classes — one  a  large  one,  of  those  organisms 
which  did  not  have  the  property  of  nitrate  reduction,  and  the  two 
smaller  classes,  in  which  were  those  which  formed  nitrites  and  those 
which  formed  ammonia. 

Table  31  shows  a  somewhat  surprising  lack  of  correlation  between 
the  formation  of  the  two  reduction  products  for  which  we  have  made 
tests.     Only  17  cultures  showed  both  nitrites  and  ammonia  in  five 


Generic  Characters  in  the  Coccaceae 


189 


or  more  of  the  10  tubes,  while  48  cuUures  formed  nitrites  alone,  and  53 
cultures  ammonia  alone,  according  to  the  same  standard.  It  seems 
improbable  that  in  the  latter  case  nitrites  had  been  formed  and 
entirely  reduced  to  ammonia.     We   are  inclined  rather  to  conclude 

TABLE  31. 
Correlation  between  Nitrite  Formation  and  Ammonia  Formation. 


Nitrites + 

Nitrites  — 

Ammonia  •+■ 

17 
Si 

382 

Ammonia — 

that  two  different  types  of  reduction  exist,  in  one  of  which  ammonia 
is  produced  directly. 

As  regards  correlation  with  other  properties,  we  have  seen  that 
the  production  of  nitrites,  and  still  more  notably  that  of  ammonia, 
is  especially  characteristic  of  the  cocci  isolated  from  the  air.  It 
is,  of  course,  possible  that  this  may  be  indirectly  connected  with 
the  fact  that  forms  which  have  survived  drying  and  dispersal  through 
the  air  must  be  particularly  well  adapted  to  conditions  which  obtain 
in  the  nitrate  solution.  A  similar  law  is  apparently  manifest  in  the 
striking  relation  to  vigor  of  surface  growth.  The  power  of  forming 
both  reduction  products  increases  progressively  with  the  richness 
of  surface  growth,  being  entirely  absent  in  the  "very  faint"  class. 
No  relation  appears  between  nitrate  reduction  and  optimum  tem- 
perature, and  the  only  other  correlation  to  be  considered  is  that 
with  gelatin  liquefaction  shown  in  Table  32. 

TABLE  32. 

Correlation  between  Nitrate  Reduction  and  Gelatin  Liquefaction. 


Gelatin  Liquefaction 

(in  cm.) 

Nitrates 
Not  Reduced 

Nitrites  Formed 

Ammonia  Formed 

JO.           

152 
131 

99 

26 
27 
17 

13 

35 

1 . 6  and  over 

27 

Table  32  shows  the  usual  large  proportion  of  organisms  which 
do  not  exert  nitrate  reduction,  but  it  may  be  noticed  that  some  30 
per  cent  of  the  liquefiers  reduce  nitrates,  against  only  20  per  cent 
of  the  non-liqucficrs.  Again,  only  6  per  cent  of  the  non-liqueticrs, 
against    16   per  cent   of  the   liquefiers,   form   ammonia. 


190  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

7.  Optimum  Temperature. 

We  have  divided  the  cocci  into  five  groups,  according  to  their 
optimum  growth  temperature.  Forty-one  cuhures  gave  "much 
better,"  and  42  "better,"  growth  at  20°;  335  developed  "equally" 
at  both  temperatures;  57  grew  "better,"  and  25  "much  better," 
at  37°. 

We  have  classed  together  the  first  two  and  last  two  groups.  In 
making  the  tables  it  was  more  convenient  to  have  fewer  groups,  and 
quite  as  accurate,  since  the  main  distinctions  (and  those  not  very 
rigid)  are  shown  in  "better  growth  at  20°"  or  "better  at  37°,"  and 
"equal"  growth  at  both  temperatures. 

We  have  already  noted  the  correlation  between  optimum  tem- 
perature and  habitat,  the  parasitic  forms  growing  best  at  37°,  and 
the  saprophytic  forms  at  20°,  when  any  difference  appears.  The 
sarcinas  belong  notably  to  the  second  class,  as  do  the  Gram-positive 
cultures.  These  are  the  only  correlations  which  have  so  far  ap- 
peared. 

We  have  been  somewhat  surprised  not  to  find  special  correlation 
between  the  optimum  temperature  for  growth  and  that  for  color 
production;  but  no  such  correlation  appears.  With  gelatin  liquefac- 
tion also  no  definite  relation  appears. 

We  have  observed  also  the  effect  of  the  body  and  room  tempera- 
ture upon  color  production,  but  without  important  results.  Of 
the  cocci  studied,  69  showed  a  very  much  higher  chromogenic  power 
at  20°  than  at  37°;  169  showed  more  color,  but  not  so  much  more, 
at  the  lower  temperature;  in  245  cases  no  difference  appeared,  while 
13  cultures  showed  more,  and  14  cultures  much  more,  pigment  at 
37°.  We  have  calculated  correlation  tables  for  all  the  various  char- 
acters studied,  but  in  no  case  did  any  constant  relation  appear, 
except,  as  noted  later,  in  connection  with  the  kind  of  chromogenesis. 

8.     Chromogenesis. 

As  noted  above,  chromogenesis  was  determined  by  matching 
the  pigment  dried  on  white  paper  against  a  color  chart  prepared 
after  a  thorough  study  of  the  colors  actually  found  among  the  cocci. 
This  chart  included  nine  hues,  designated  by  Roman  numerals, 
corresponding    to    the    pigments    noted    below    the    figure.     Under 


Generic  Characters  in  the  Coccaceae  191 

each  hue  were  nine  different  chromas,  indicated  by  Arabic  numerals, 
each  figure  indicating  the  number  of  washes  of  pure  color  added 
to  obtain  the  particular  chroma. 

The  distribution  of  the  organisms  studied  under  these  different 
colors  is  indicated  in  Fig.  3,  where  the  vertical  columns  indicate 
the  hues  from  I  (white),  through  the  yellows  (II-IV),  the  oranges 
(V  and  VI)  to  the  reds  (VII-IX),  and  the  horizontal  columns  the 
successive  chromas. 

On  inspection  of  this  chart,  bearing  in  mind  the  colors  signified, 
there  appear  at  once  four  modes  -  one  occurring  in  each  chief  color. 

That  for  the  white  falls  at  I,,  for  the  yellows  at  IV,,  for  the  oranges 
at  VIg,  and  for  the  reds  at  Vllg.  These  are  not,  of  course,  the 
points  of  intcnsest  color,  but  of  the  most  concentrated  distribution. 
The  evident  clustering  of  the  individuals  around  a  mode,  and  the 
consequent  falling-away  of  the  numbers  between  the  modes,  sug- 
gest a  variation  from  an  ancestral  center.  Like  most  living  things 
governed  by  an  evolutionary  law  of  gradual  change,  the  hues  grade 
so  gently  into  each  other  that  the  exact  placing  of  lines  of  division 
must  be  arbitrary.  We  have,  however,  assumed  four  divisions  as  a 
basis  for  our  work,  and  separated  them  at  the  lowest  points  be- 
tween the  modes,  as  shown  by  the  heavy  black  lines  in  the  chart, 
which  divide  the  group  of  bacteria  producing  a  white  pigment  from 
that  which  produces  a  yellow,  the  yellow  from  the  orange,  and  the 
orange  from  the  red.  The  striking  correlations  obtained  between 
chromogenesis  and  other  properties  have  convinced  us  that  this  divi- 
sion was  a  sound  and  natural  one.  It  should  be  noted,  however, 
that  the  division  of  the  "white"  chromogens  includes  two  sub- 
groups— the  true  white  pigment-formers  and  the  forms  which  pro- 
duce such  a  faint  surface  growth  that  no  distinct  color  is  apparent. 

We  have  omitted  the  consideration  of  chromogenesis  from  our 
correlation  tables,  except  that  for  habitat,  preferring  to  consider 
all  chromogenic  relations  under  one  head.  It  will  appear,  on  inspec- 
tion of  the  following  tables,  that  this  character  is  really  the  key 
by  which  most  of  the  other  correlations  may  be  explained,  and  is 
perhaps  the  most  important  single  factor  in  the  systematic  grouping 
of   the    Coccacese. 

It  was  shown  under  "Habitat"  that  the  white  and  orange  chrome- 


19: 


C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 


gens  were  chiefly  parasites,  the  yellow  and  red  chromogens  chiefly 
saprophytic  forms.  The  same  distinction  is  shown  in  Table  ^t,  with 
regard  to  cell-grouping.     The  white  and  orange  cocci  only  rarely, 


TABLE  33. 
Correlation  between  Chromogenesis  and  Cell-Gro0ping. 

Cell-Grouping 

White 

Yellow 

Orange 

Red 

Irregular  Groups  and  Chains 

Packets 

33 

7 

134 
120 

163 
18 

IS 
10 

the  latter  very  rarely,  show  packets.     The  yellow  and  red  forms, 
on  the  other  hand,  show  the  sarcinae-grouping  almost  half  the  time. 

TABLE  34. 
Correlation  between  Chromogenesis  and  Gram  Stain. 


Gram  Stain 

White 

Yellow 

Orange 

Red 

6 

9 

25 

lOQ 
84 
61 

IS 

46 

120 

15 
7 
3 

± 

+ 

The  reaction  to  the  Gram  stain  exhibits  a  still  more  perfect 
correlation.  Among  the  whites  and  oranges  (the  parasitic  forms) 
positive  Gram  reactions  predominate,  and  negative  ones  are  rare. 
Among  the  saprophytic  yellows  and  reds  conditions  are  symmetri- 
cally reversed. 

TABLE  35. 
Correlation  between  Chromogenesis  and  Surface  Growth. 


Surface  Growth 

Very  faint 

Meager 

Good 

Abundant 

Very  heavy 


White 

Yellow 

Orange 

14 

3 

2 

3 

6 

12 

7 

100 

107 

13 

95 

58 

3 

SO 

2 

Red 


o 
o 

I 

24 

o 


A  comparison  of  the  general  vigor  of  growth  shows  that  each 
color  has  its  own  relation.  Among  the  white  forms,  two  maxima 
appear,  one  under  "very  faint"  growth  and  one  under  "abundant" 
growth.  This  is  because  this  group  is  a  compound  one,  including 
forms  which  give  a  really  w^hite  growth  abundant  in  amount,  and 
the  feebly  growing  streptococci  which  are  classed  here,  although 
they  produce  no  pigment  at  all.     The  yellow  and  orange  chromo- 


Generic  Characters  in  the  Coccaceae 


193 


gens  show  maxima  under  the  "good"  growth,  almost  all  the  "very 
abundant"  growths  belonging  to  the  former  class.  The  red  forms 
are  almost  all  of  one  type — the  "abundant." 

TABLE  36. 
Correlation  between  Chromocenesis  and  Dextrose  Fermentation. 


Acid  Produced  (Per  Cent  Normal) 

0.0  and  alkaline 

o. i-o. 2  

0.3-0.6 

o . 7-2 .0 

Over  2.0 


White 


S 
7 
5 
15 
8 


YeUow 


04 
72 
SO 
iS 
3 


Orange 


7 
24 

02 

S3 
S 


Red 


TABLE  37. 
Correlation  between  Chrouogenesis  and  Lactose  Fermentation. 


Add  Produced  (Per  Cent  Normal) 

White 

Yellow 

Orange 

Red 

-0. 2  and  more  alkaline 

3 
8 

12 
6 

It 

33 
141 

59 

18 

3 

9 
39 

64 

63 

6 

S 
16 

—0 . 1  and  0.0 

0.1-0.4 

3 

0 .  ^— I  .A 

The  correlations  between  chromogcnesis  and  the  fermentation 
of  the  sugars  arc  singularly  perfect.  The  white  forms  in  each  case 
show  two  maxima,  one  corresponding  to  the  true  white  chromo- 
gens,  the  second,  at  a  higher  acidity,  to  the  colorless  streptococci. 
The  latter  include  a  majority  of  the  strongest  acid-producers  in 
each  case.  The  other  types  show  for  each  sugar  a  regular  and  char- 
acteristic curve,  the  elements  from  which  the  complex  curve  in 
Fig.  2  is  made.  The  yellow  forms  show  for  each  sugar  a  mode  at 
the  neutral  point.  The  orange  chromogens,  on  the  other  hand, 
are  most  abundant  at  an  intermediate  grade  of  acidity,  most  of 
them  producing  0.3-0.6  per  cent  acidity  in  dextrose  broth,  and 
0.1-0.4  per  cent  in  lactose  broth.  The  red  forms  show  the  same 
relation  as  the  orange  forms  toward  dextrose,  while  in  lactose  broth 
they  resemble  the  yellow  chromogens,  producing  in  most  cases  no 
change  of  reaction. 

TABLE  38. 
Correlation  between  Chromogenesis  and  Nitrate  Reddction. 


No  reduction 

Nitrites  produced  . 
Ammonia  produced 


White 


35 
3 

2 


Yellow 


197 
30 
37 


Orange 


137 
as 
26 


Red 


«3 
12 

o 


194 


C.-E,  A.  WiNSLow  AND  Anne  F.  Rogers 


With  regard  to  the  reduction  of  nitrates,  the  white  and  colorless 
forms  show  generally  negative  results.  Nitrites  are  produced  by 
one  in  lo  of  the  yellows,  a  slightly  higher  fraction  of  the  orange 
forms,  and  by  half  the  red-pigment-producers.  Ammonia  produc- 
tion, on  the  other  hand,  appears  in  one  in  eight  of  the  yellows,  one 
in  I o  of  the  orange  forms,  and  not  at  all  among  the  reds. 

TABLE  39. 
Correlation  between  Chromogenesis  and  Optimum  Temperature  for  Growth. 


Optimum  Temperature 

20° 

20°  or  37° 

37° 


WUte 


Yellow 


Orange 


Red 


4 

28 

8 


66 

156 

32 


13 

126 

42 


o 

25 

o 


Excluding  the  majority  of  forms  which  grow  equally  at  either 
temperature,  it  appears  that,  among  the  white  and  orange  forms, 
most  of  those  which  exhibit  any  preference  grow  best  at  37°,  while 
among  the  yellows  20°  is  more  often  the  optimum.  These  results 
accord  with  the  habitats,  respectively  parasitic  and  saprophytic, 
of  the  two  classes. 

TABLE  40. 
Correlation  between  Chromogenesis  and  Optimum  Temperature  for  Color  Formation. 


Color  Production 

White 

YeUow 

Orange 

Red 

Better  at  20° 

2 
38 

90 
164 

125 
56 

21 

Equal  at  20*^  and  37*^  .          

4 

It  appears  from  Table  40  that  temperature  differences  affect 
the  production  of  orange  pigment  much  more  than  that  of  yellow 
and  that  the  body  temperature  interferes  with  red  chromogenesis 
most  of  all. 

TABLE  41. 
Correlation  between  Chromogenesis  and  Gelatin'Liquefaction. 


Gelatin  Liquefaction,  cm. 

WTiite 

YeUow 

Orange 

Red 

0.0 

0.1-1.5 

1 . 6  and  over 

27 
8 
5 

83 
126 

45 

55 
39 
87 

21 

4 
0 

The  licjuefaction  of  gelatin  presents  another  close  correlation 
with  pigment  production.  The  white  and  red  forms  are  almost 
all  non-liquefiers,  the  yellow  cocci  show  a  maximum  among  the 


Generic  Characters  in  the  Coccaceae 


195 


moderate     liquefiers,    and     the     orange     chromogens     exhibit     the 
peptonizing  power  to  a  high  degree. 

To  sum  up,  the  cocci  show  four  (or  five)  distinct  groups  according 
to  their  pigment  production,  each  group  being  marked  by  a  number 
of  other  correlated  characters.  The  "white"  forms  rarely  show 
packets,  generally  stain  by  Gram,  fail  to  reduce  nitrates,  grow  well 
at  37°,  and  usually  fail  to  licjucfy  gelatin.  They  include  two  sub- 
groups— the  feebly  growing,  strongly  acid-producing  forms,  which 
are  really  colorless,  not  white,  and  the  white-pigment-producers, 
which  grow  abundantly  and  produce  only  a  slight  amount  of  acid.  The 
"yellow"  chromogens  frequently  show  packets,  are  usually  Gram- 
negative,  give  a  good  to  a  very  heavy  surface  growth,  produce  little  or 
no  acid,  occasionally  form  nitrites  or  ammonia  in  nitrate  solution, 
grow  well  at  20°,  and  show  a  moderate  liquefaction  of  gelatin.  The 
"orange "-pigment-formers  are  very  rarely  packets,  stain  well  by 
Gram,  form  good  surface  growths,  produce  a  moderate  acidity  in 
sugar  broth,  occasionally  reduce  nitrates  to  nitrites  or  ammonia,  grow 
well,  but  with  poor  pigment  production,  at  37°,  and  generally  produce 
a  considerable  liquefaction  of  gelatin.  The  red-pigment-producers 
are  often  packets,  generally  Gram-negative,  grow  abundantly,  ferment 
dextrose  but  not  lactose,  form  nitrites,  but  not  ammonia,  in  nitrate 
solution,  grow  well  at  20°  or  37°,  producing  less  pigment  in  the  latter 
case,  and  generally  fail  to  liquefy  gelatin. 

9.  Gelatin  Liquefaction. 

In  the  routine  determination  of  gelatin  liquefaction  we  have 
used  only  one  tube  for  each  culture.  Duplicate  determinations 
were  made  on  79  cultures,  from  which  it  appeared  that  the  probable 
error  of  a  single  observation  is  only  ±0.12  cm.;  so  that  our  method 
was  sufficiently  accurate. 

Of  the  cultures  observed,  186  failed  to  liquefy  gelatin,  and  the 
distribution  of  the  other   314  cultures    according  to  the  amount  of 


TABLE  42. 
Gelatin  Liquefaction. 


Depth  in  cm 

Number  of  cultures 


0. i-o. 5 
33 

0.6-1 
76 

1.1-1.5 
68 

1.6-a.o 
48 

2 . i-a . 5 
44 

7 . 6-3 . 0 
29 

3   1-3.S 
«3 

over  3.S 
3 


196  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

liquefaction  after  four  weeks  is  shown  in  Table  42.  Fig.  4  shows 
graphically  the  skew  curve  plotted  from  these  data. 

There  is,  as  would  be  expected,  a  gradual  falling-away  toward 
the  highest  amounts  of  liquefaction,  and  the  abrupt  downward 
falling  of  the  curve  toward  the  non-liquefiers  at  o  is  extremely  sig- 
nificant as  indicating  a  sharp  difference  between  the  two  groups. 
If  it  had  been  practicable  to  plot  the  non-liquefiers  on  this  figure, 
the  curve  would  have  gone  up  at  an  acute  angle  more  than  twice 
as  high  as  that  of  the  mode  of  the  liquefiers.  This  angle  divides 
with  more  than  usual  definiteness  those  organisms  which  liquefy 
from  those  which  do  not  liquefy  gelatin. 

The  correlations  of  gelatin  hquefaction  with  other  properties 
have  been  already  considered.  We  have  found  that  a  high  pepton- 
izing power  is  rare  among  the  earth  and  water  cocci  and  the  sarcinae. 
It  is  most  frequently  associated  with  the  smaller  individual  cells 
among  the  non-packet-formers.  It  is  absent  or  very  rare  in  the  cocci 
which  show  only  faint  surface  growths.  Finally,  it  appears  that 
the  white,  colorless  forms  which  produce  high  acidities,  as  well 
as  the  red  chromogens,  are  non-liquefiers.  The  yellow  cocci  which 
produce  little  acid  are  moderately  active  liquefiers,  and  the  orange 
forms  with  a  moderate  acid  production  show  the  highest  pepton- 
izing power. 

IV.    CONCLUSIONS  FROM  THE  INVESTIGATION. 

I.    Foundation  of  Subfamilies  and  Genera  among  the  Cocci. 

The  extreme  variability  of  the  cocci  has  appeared  with  great 
clearness  in  the  present  study.  Almost  every  one  of  the  characters 
measured  shows  a  wide  range  of  fluctuations.  In  view  of  the  gen- 
eral laws  of  variation,  the  absence  of  sexual  reproduction,  and  the 
susceptibility  of  the  bacteria  to  the  direct  influence  of  the  environ- 
ment, this  is  precisely  what  should  be  expected.  It  makes  it,  however, 
clearly  impossible  to  draw  sharp  and  arbitrar)^  lines  for  any  single 
character  by  which  individual  organisms  can  be  naturally  classified. 

If,  on  the  other  hand,  we  examine  a  series  of  individuals  with 
the  idea  of  discerning  central  types  about  which  they  vary,  the  prob- 
lem begins  to  solve  itself,  since  such  types  are  easily  apparent.  Cer- 
tain organisms  tend  to  show  the  packet  grouping — some  invariably 


Generic  Characters  in  the  Coccaceae  197 

in  every  aggregate,  some  less  constantly.  Other  organisms  never 
show  packets,  or  only  very  rarely.  Some  cocci  always  stain,  and 
some  always  decolorize,  by  Gram,  while  intermediate  forms  tend 
more  or  less  strongly  toward  either  type.  In  surface  growth  two 
distinct  types,  the  faint  to  meager  and  the  abundant  to  very  heavy, 
are  manifest.  In  acid  production  there  appear  to  be  three  centers 
of  distribution  corresponding  to  organisms  which  fail  to  ferment, 
those  which  ferment  slightly,  and  those  which  produce  large  amounts 
of  acid.  In  relation  to  nitrate  reduction,  three  types  appear,  accord- 
ing as  the  cocci  fail  to  reduce,  or  form  nitrites  or  ammonia.  On 
gelatin  the  organisms  studied  group  themselves  either  as  liquefiers 
or  as  non-liquefiers,  and  in  color  production  four  distinct  centers 
appear,  in  which  the  pigment  is  white,  yellow,  orange,  or  red. 

Our  estimate  of  the  value  of  these  type-centers  is  greatly  in- 
creased when  we  find  that  the  central  points  for  the  different  characters 
do  not  vary  independently,  but  are  correlated  together  to  a  remark- 
able degree.  Again,  we  should  expect,  and  we  actually  find,  in  some 
cases,  the  correlation  of  single  characters  varying,  those  properties 
generally  correlated  appearing  in  certain  organisms  in  exceptional 
combinations.  If,  however,  we  consider,  not  the  single  character — 
not  the  individual  organism — but  the  aggregate  of  the  correlations  of 
various  properties  as  manifested  in  a  considerable  series  of  indi- 
viduals, certain  well-defined  systematic  units  appear,  marked  by  the 
association  of  a  number  of  independent  characteristics.  Such  an 
association  can  be  explained  only  on  the  ground  of  relationship,  and 
the  types  marked  by  the  simultaneous  occurrence  of  a  number  of 
properties  may  rightly  be  taken  as  the  centers  from  which  other, 
more  aberrant  individuals  have  varied. 

The  fact  that  correlation  exists  shows  that,  on  the  average,  the 
fluctuations  of  these  characters  do  not  occur  independently,  but 
are  so  closely  bound  up  with  those  of  other  properties  as  to  vary 
together  with  them.  This  may  be  because  the  selective  action 
of  the  environment  produces  a  parallel  change  in  each,  or  because 
the  two  characters  are  so  closely  bound  together,  in  the  physiological 
balance  of  the  organism,  that  a  change  in  one  leads  to  a  corresponding 
variation  in  the  other.  In  either  event  it  is  clear  that  the  larger 
systematic  units  (families  or  genera)  must  be  marked  by  these  pro- 


198  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

found  modifications  of  the  whole  center  of  gravity  of  the  organism, 
and  the  smaller  groups  by  those  characters  which,  though  perhaps 
showing  sharper  individual  differences,  vary  by  themselves  without 
affecting  any  other  properties. 

Our  object  therefore  has  been,  not  to  establish  arbitrary  boundary 
lines,  but  to  discover  existing  natural  types  distinguished  by  the 
association  of  independent  characters.  In  such  a  task  it  is  obvious 
that  those  characters  are  most  important  which  show  the  most  marked 
correlations.  What  these  characters  are  must  be  determined  by  study 
in  each  particular  group.  Chromogenesis  or  gelatin  liquefaction 
may  be  of  generic  value  in  one  family,  or  may  mark  only  varieties 
in  another,  as  it  is,  or  is  not,  correlated  with  a  number  of  other 
properties.  In  the  Coccaceae,  for  example,  the  liquefaction  of  gelatin 
and  the  reduction  of  nitrates  appear,  when  judged  by  this  standard, 
to  be  of  less  importance  than  most  of  the  properties  studied.  In  some 
cases  they  appear  to  be  significant,  but,  in  most  of  the  groups  indi- 
cated, liquefying  and  non-liquefying  forms,  and  reducing  and  non- 
reducing  forms,  run  parallel.  Distinctions  based  on  such  a  single 
character  alone  may  have  specific,  but  certainly  not  generic,  value. 
On  the  other  hand,  we  have  been  somewhat  surprised  to  find  that 
such  apparently  fluctuating  characters  as  chromogenesis  and  the 
reaction  to  the  Gram  stain  are  strongly  correlated  with  a  number 
of  other  properties. 

A  general  survey  of  the  whole  field  of  variation  among  the  Cocca- 
ceae indicates  clearly  the  existence  of  two  main  sets  of  correlated 
characters,  corresponding  to  the  subfamilies  which  we  have  suggested 
in  a  previous  communication  (Winslow  and  Rogers,  1905).  Habitat, 
morphology,  staining  reactions,  surface  growth,  acid  production, 
optimum  temperature,  and  chromogenesis,  all  vary  simultaneously 
in  one  or  the  other  of  two  directions,  defining  the  two  subfamilies 
Paracoccaceae  and  Metacoccaceae.  The  first  group,  comprising  most 
of  the  forms  from  the  body,  shows,  as  a  rule,  chains  and  irregular 
cell-grouping,  stains  by  Gram,  yields  a  meager  or  only  fair  surface 
growth,  forms  acid  in  carbohydrates,  and  produces  no  pigment,  or  a 
white  or  an  orange  one.  The  other  group,  from  earth  and  water  for 
the  most  part,  often  shows  packets,  decolorizes  by  Gram,  grows  well 
on  artificial  media,  fails  to  ferment  carbohydrates,  and  produces  a 


Generic  Characters  in  the  Coccaceae  199 

yellow  or  red  pigment.  It  must  always  be  remtmbLrtd  that  each 
character  may  occasionally  be  found  in  the  group  where  it  usually  does 
not  occur;  but  the  association  of  these  properties  in  the  vast  majority 
of  cases  is  very  strong.  We  desire  to  extend  our  earlier  definitions 
of  the  two  subfamilies  by  including  the  Gram  reaction  and  chromo- 
genesis;  and  the  subfamilies  as  thus  modified  will  be  defined  at  the 
end  of  this  communication.  It  is  a  striking  fact  that  these  two  chief 
divisions  among  the  Coccaceae  correspond  to  the  two  markedly  dilTerent 
environments  which  exist  in  nature,  the  body  of  higher  organisms, 
and  the  outer  world.  A  close  correspondence  with  environmental 
conditions  should  naturally  be  expected  among  such  simple  asexual 
organisms  as  the  bacteria,  and  it  increases  our  confidence  in  the 
reality  of  the  groups  established  below  to  find  each  of  thim  localized 
so  sharply  in  one  or  other  of  the  two  main  environments. 

Under  the  subfamilies  we  find  a  second  grade  of  group-individu- 
ality, marked  by  the  association  of  a  smaller  number  of  characters 
than  the  subfamilies,  but  still  defined  by  the  correlation  of  several 
independent  properties.  Here  morphology,  surface  growth,  and 
chrcmogencsis  appear  to  be  of  greatest  importance,  acid  production, 
gelatin  liquefaction,  and  nitrate  reduction  having  special  significance 
in  certain  cases.  Five  distinct  types  have  appeared  with  consider- 
able clearness  in  the  present  study.  It  must  be  remembered  that 
the  fundamental  correlations  which  revealed  these  groups  were 
derived  in  an  entirely  impersonal  way  by  measurements,  made  on 
each  character  independently,  generally  by  different  observers, 
and  always  without  knowledge  of  the  identity  of  the  organism. 
When  individual  races  are  considered,  it  is  possible,  by  transferring 
a  few  cultures  on  the  border-line  in  a  single  character,  to  show  that 
the  correlations  are  really  closer  than  have  appeared  above. 

By  this  process  we  have  attempted  to  group  our  500  cultures 
under  the  five  subdivisions  suggested  by  the  correlation  tables,  and 
have  found  the  results  so  satisfactory  as  to  confirm  our  confidence 
in  their  reahty  as  natural  groups.  It  seems  to  us  that  these  groups 
are  of  such  importance  as  to  deserve  generic  rank.  Within  each 
there  is  ample  room  for  the  establishment  of  such  a  reasonable 
number  of  species  as  detailed  study  may  warrant.  Good  genera 
must  first  be  recognized,  however.     It   is  time  that  bacteriologists 


200  C.-E.  A.  WiNSLOw  AND  Anne  F.  Rogers 

were  relieved  of  such  vast  and  unwieldy  and  meaningless  genera 
as  now  burden  the  science. 

The  first  of  these  groups  centers  about  a  type  of  organism  char- 
acterized by  the  following  properties:  it  is  parasitic  in  habit  and 
grows  in  irregular  groups,  often  in  chains,  never  in  packets;  it  stains 
by  Gram;  it  grows  in  a  thin  film  on  the  surface  of  agar;  it  ferments 
both  lactose  and  dextrose  with  the  production  of  a  large  amount  of 
acid;  it  fails  to  reduce  nitrates  or  hquefy  gelatin,  grows  best  at  37°, 
and  forms  no  appreciable  amount  of  pigm.ent.  This  corresponds 
to  the  genus  Streptococcus  (Billroth)  W.  and  R.,  as  previously  char- 
acterized. We  desire  to  add  to  our  previous  conception  of  the  group 
the  positive  reaction  to  the  Gram  stain  and  the  general  failure  to 
act  on  gelatin  or  nitrates.  It  must  always  be  remembered  that  this 
genus  is  defined,  not  on  morphology  alone,  although  its  members 
generally  do  show  long  chains  in  broth,  but  by  the  general  complex 
of  all  its  characters.  Individual  cultures  vary  from  the  type  in 
some  respects,  as  must  all  aggregates  of  organisms  composed  of 
such  varying  stuff  as  hving  protoplasm. 

Of  our  500  cultures  18  fall  into  the  genus  Streptococcus.  All 
show  the  typical  morphology  (groups  and  long  and  short  chains)  and 
typical  surface  growth.  None  liquefy  gelatin  or  reduce  nitrates.  Of 
our  18  cultures,  15  were  from  the  body  and  three  from  polluted  water. 
In  relation  to  the  Gram  stain,  11  cultures  showed  positive  tests,  on 
both  trials,  and  only  two  a  negative  test,  five  being  variable.  Of 
the  18  cultures,  nine  showed  very  high  acidities,  over  2  per  cent 
normal,  in  both  acids,  some  ranging  as  high  as  8  to  9  per  cent.  The 
average  value  for  the  whole  genus  is,  for  dextrose  2.6  per  cent, 
and  for  lactose  i .  7  per  cent.  It  is  interesting  to  notice  that  those 
cultures  which  yield  lower  acidities  are  also  the  ones  which  give 
the  negative  or  variable  Gram  reactions.  Our  forms  therefore  seem 
to  fall  into  two  species,  10  of  them  belonging  to  the  Str.  erysipelatos, 
showing  the  very  high  acidities  and  the  positive  Gram  reaction; 
the  other  eight  differing  in  both  these  characters. 

The  second  of  our  five  groups  is  marked  by  a  correlation  of  char- 
acters, of  which  the  most  obvious  is  the  production  of  an  orange 
pigment.  In  our  previous  communication  we  were  unable  to  dis- 
tinguish, from  the  literature  alone,  any  sharp  line  between  the  orange 


Generic  Characters  in  the  Coccaceae  201 

and  yellow  chromogens,  and  included  them  both  under  the  genus 
Micrococcus.  Fig.  3  makes  it  clear,  however,  that  two  distinct 
centers  of  variation  exist,  one  in  the  orange  and  one  in  the  yellow, 
and  our  correlation  tables  show  that  the  two  types  of  organisms  arc 
so  radically  different  in  every  character  as  to  demand  their  separa- 
tion into  distinct  genera.  Furthermore,  it  is  evident  that  the  orange 
chromogens  belong  with  the  parasitic  Paracoccaceae,  and  the  yellow 
forms  with  the  Metacoccaceae.  Nothing  could  show  more  clearly 
how  necessary  it  is  to  make  a  comparative  study  of  a  large  series  of 
organisms  in  order  to  discern  the  true  relationships  of  the  bacteria. 

For  this  new  genus  we  suggest  the  name  Aurococcus,  as  indicat- 
ing the  orange  color,  which  is  its  most  obvious  characteristic.  Its 
type-form  is  found  on  or  in  the  plant  or  animal  body.  It  occurs 
in  groups  and  short  chains,  stains  by  Gram,  and  produces  a  good, 
but  not  heavy,  surface  growth  of  an  orange  color.  It  ferments 
dextrose  and  lactose,  producing  an  acidity  generally  between  o .  5 
and  I.  It  grows  well,  but  produces  less  pigment  at  37°.  It  may 
or  may  not  reduce  nitrates  and  liquefy  gelatin.  When  it  does  liquefy 
gelatin,  it  does  so  rather  actively. 

Of  the  158  cultures  in  this  group,  all  show  a  good,  but  not  very 
abundant,  growth  of  an  orange  color;  116  were  obtained  from  the 
body  and  30  from  the  air,  only  12  having  a  saprophytic  origin;  147 
show  groups  and  short  chains,  but  no  packets;  and  11  occasionally 
give  the  sarcina  grouping.  Of  the  158  cultures,  107  show  a  positive 
Gram  reaction,  and  only  nine  a  consistently  negative  one.  The  aver- 
age acidity  in  dextrose  for  the  whole  group  is  0.7  per  cent  normal, 
and  for  lactose  0.4  per  cent  normal.  Of  the  158  cultures  only 
six  form  less  than  o .  2  per  cent  acid,  and  1 7  more  than  i  per  cent 
acid,  in  dextrose.  In  lactose  there  is  more  variation;  53  cultures 
give  less  than  0.2  per  cent,  and  11  more  than  i  per  cent,  acid. 
Of  the  cultures,  31  reduce  nitrates,  and  102  liquefy  gelatin  to  an 
average  depth  of  2.2  cm. — a  very  high  value;  while  56  organisms 
fail  to  hquefy.  The  type-form  of  this  genus  is  the  commonest  pyo- 
genic organism,  the  M.  aureus  of  Rosenbach.  The  non-liquefying 
forms,  those  which  reduce  nitrates,  and  those  which  produce  more 
or  less  acid  than  is  common  in  the  genus,  may  later  be  set  up  as 
separate  species. 


202  C.-E.  A,  WiNSLOW    AND    AnNE    F.  RoGERS 

The  third  of  our  types,  like  the  second,  has  not  previously  been 
distinguished  from  the  genus  Micrococcus;  it  appears,  however, 
to  shov^r  its  own  definite  individuality,  and  to  belong  with  the  Para- 
coccaceae,  although  it  approaches  the  saprophytic  cocci  in  certain 
characters.  We  suggest  the  name  Alhococcus  for  this  genus,  which 
includes  those  organisms  of  which  M.  pyogenes  (Ros.)  Mig.  is  a 
type.  They  produce  a  more  vigorous  surface  growth  than  the  strep- 
tococci, with  a  clear  white  pigment,  and  ferment  carbohydrates, 
producing  a  fair  amount  of  acid.  They  are  also  distinguished  from 
the  Metacoccaceae  by  the  general  tendency  of  their  morphology  and 
staining  reactions,  and  by  their  habitat.  In  our  series  we  have  23 
cultures  of  this  type.  All  without  exception  were  obtained  from  the 
body  or  from  the  air,  none  from  water  or  earth.  All  without  excep- 
tion show  a  good  surface  growth,  white  pigment,  and  division  into 
groups  and  rarely  chains,  but  never  packets.  Sixteen  were  uni- 
formly Gram  positive  and  only  two  uniformly  Gram  negative.  The 
average  acidity  in  dextrose  broth  was  0.7  per  cent  normal,  and 
in  lactose  broth  o .  5  per  cent  normal.  Only  three  cultures  showed 
an  acidity  lower  than  o .  2  per  cent,  and  only  one  culture  an  acidity 
over  1 . 5  per  cent  in  dextrose.  Lactose  results,  as  usual,  were  more 
variable,  nine  cultures  falling  below  o .  2  per  cent  acid,  and  one  above 
1.5  per  cent.  Nitrates  were  reduced  by  three  cultures  and  gelatin 
liquefied  by  14.  The  four  species  which  we  have  previously  call- 
ed M.  pyogenes  (Ros.)  Mig.,  M.  rhenanus  Mig.,  M.  candicans 
Fliigge,  and  M.  canescens  Mig.,  should  belong  to  this  new  genus, 
being  distinguished,  as  before,  by  their  relation  to  acid  and 
gelatin.  The  reduction  of  nitrates  may  furnish  a  basis  for  the 
establishment  of  other  species. 

The  fourth  of  the  general  groups  which  have  appeared  in  this 
study  is  the  group  of  the  yellow  pigment  formers,  of  which  M.  lute- 
us  and  Sarcina  ventriculi  are  typical — a  group  which  differs  in  almost 
all  its  properties  from  those  previously  considered.  Organisms  of 
this  type  are  found  mainly  in  earth  and  water  rather  than  on  or  in 
the  animal  body.  They  give  abundant,  to  very  heavy,  growths 
of  a  yellow  color.  They  frequently  occur  in  packets,  generally  decol- 
orize by  Gram,  and  fail  to  ferment  sugars  or  ferment  them  only 
slightly.     They  may  or  may  not  liquefy  gelatin  or  reduce  nitrates. 


Generic  Characters  in  the  Coccaceae 


203 


Of  our  cultures  262  fall  under  this  head,  195  of  them  coming 
from  water,  earth,  or  air,  and  only  64  from  the  body;  200  are  uni- 
formly or  at  times  Gram  negative,  and  only  62  uniformly  Gram  pos- 
itive. The  average  acidity  produced  in  dextrose  broth  is  only 
0.2  per  cent  normal,  and  in  lactose  broth  o.i  per  cent  normal. 
Of  the  262  cultures  only  t,t,  give  over  o . 5  per  cent  acid  in  dextrose 
broth,  and  only  7  over  o .  5  per  cent  acid  in  lactose  broth ;  59  of  the 
cultures  reduce  nitrates;  85  fail  to  act  on  gelatin,  and  177  liquefy  it, 
producing  an  average  liquefaction  of  1.2  cm.,  scarcely  more  than 
half  the  value  in  the  genus  Aurococcus. 

This  group  divides  itself,  according  to  cell-grouping,  into  two 
nearly  equal  divisions — those  which  form  packets  and  those  which 
do  not;  136  belong  to  the  former  class,  and  126  to  the  latter.  In 
habitat,  in  Gram  staining,  and  in  relation  to  carbohydrates  and 
gelatin  both  classes  are  entirely  parallel.  The  genera  Micrococcus 
and  Sarcina  are,  however,  so  firmly  established  in  common  usage 
that  it  would  require  very  strong  evidence  of  identity  to  warrant 
dropping  either  of  them.  It  seems  best,  therefore,  to  recognize 
the  single  character  of  cell-grouping  as  having  generic  value  in  this 
case,  otherwise  defining  the  two  genera  by  the  same  characteristics. 
Under  Micrococcus  will  come  M.  orbicularis  Ravenel,  M.  luteus 
(Schroter)  Cohn,  and  M.  ochraceus  Rosenthal;  under  Sarcina,  St. 
subflava  Ravenel  and  S.  venlriculi  Goodsir. 

The  fifth  and  last  of  our  general  types  includes  the  sharply  marked 
one  of  the  red  chromogens.  These  are  entirely  saprophytic  forms 
which  produce  abundant  surface  growths  of  a  red  color.  They 
may  or  may  not  show  packets,  are  generally  Gram  negative,  very 
rarely  liquefy  gelatin  or  ferment  carbohydrates,  and  frequently 
reduce  nitrates  to  nitrites,  but  apparently  not  to  ammonia.  This 
is  the  first  case  in  which  we  have  found  the  action  upon  nitrates 
markedly  correlated  with  other  characters. 

Twenty-five  of  our  cultures  fall  under  this  general  type.  All 
but  one  come  from  earth,  water,  or  air.  Only  three  are  uniformly 
positive  to  Gram,  while  15  are  uniformly  negative.  Four  of  the  25 
cultures  liquefy  gelatin,  and  14  reduce  nitrates  to  nitrites.  The 
average  acidity  in  dextrose  broth  is  0.4  per  cent  normal,  and  in 
lactose  the  average  reaction  is  neutral.  One  culture  in  lactose 
and  four  in  dextrose  show  an  acidity  over  0.5  per  cent. 


204 


C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 


Here  again  the  packets  (lo  in  number)  and  the  other  forms  (15 
in  number)  are  exactly  parallel.  Both  show  the  same  range  of  acid- 
ities and  the  same  peculiar  relation  to  nitrate  reduction.  It  seems 
quite  clear  that  in  this  case  the  single  character  of  packet  formation 
ought  not  to  be  made  the  basis  of  a  distinct  genus.  We  have  recog- 
nized among  the  yellow  forms  the  two  genera  Micrococcus  and  Sar- 
cina out  of  deference  to  custom,  which  must  always  play  an  important 
part  in  terminology.  In  separating  the  red  forms  from  these  two 
old  genera,  however,  it  seems  an  unreasonable  recognition  of  a  false 
distinction  to  form  two  new  ones  on  the  single  character  of  cell- 
grouping  alone.  We  desire,  therefore,  to  include  all  the  red-pigment 
forms,  characterized  by  the  properties  noted  above,  under  one  new 
genus,  to  be  called  Rhodococcus.  M.  cinabareus  Fliigge,  S.  rosacea 
Lindner,  and  5.  incarnata  Gruber  will  all  belong  here.  Four- 
teen doubtful  cultures  are  for  the  present  omitted  from  this  generic 
classification. 

It  remains  only  to  summarize  the  characteristics  of  the  six  genera 
studied  in  this  investigation  in  tabular  form,  and  then  to  present 
a  systematic  statement  of  the  main  divisions  of  the  Coccaceae.  It 
must  be  remembered  that  Diplococcus  and  Ascococcus  have  not 
been  included  in  the  present  research  and  are  defined  solely  from  the 
the   literature. 

TABLE  43. 
Characters  of  Certain  Genera  of  the  Coccace^. 


Genus 

Cell-Grcuping   (Per 
Cent   0     Packet- 
Formers) 

s 

u 

was 
Si 
0 

Surface 
Grcwth 

Average   Acidity    in 
Dextrose  (Per  Cent 

Normal) 

Average   Acidity   in 
Lactose  (Per  Cent 
Normal) 

Nitrate     Reduction 
(Per  Cent  of   Re- 
ducers) 

Chromo- 
genesis 

s 

u 

4.1    CJ 

•s-5 

■z  0 
0 

Liquefaction  (Aver- 
age   Liquefaction 
cm.) 

Streptococcus 

A  urococcus 

A  Ibococcus 

Micrococcus 

Sarcina 

Rhodococcus 

83 
76 
48 
27 
22 
4 

0 
7 
0 
0 

ICO 

40 

61 
68 
70 
25 
23 
12 

Faint 

Good 

Abundant .... 
Good  to  Abun. 
Good  to  Abun. 
Abundant  .... 

2.6 
0.7 
0-7 
0-3 
0.2 
0.4 

1-7 
0.4 
o.S 
3.1 
0. 1 
0.0 

0 
21 

13 
27 
18 
S6 

Orange. 
White . . 
Yellow. 
Yellow. 
Red  .  . . 

0 
6s 
61 
68 
67 
16 

2.2 
I .  I 
1 .2 
1.2 
0.7 

♦Body  alone;  air,  source  of  many  others.     In  Albococcus  none  from  water  or  earth. 

2.  Systematic  Summary. 
Family    Coccaceae:  Vegetative  cells  spherical. 
Subfamily    i    Paracoccaceae    (Winslow    and    Rogers):  Parasites 
(thriving  only  or  best,  on  or  in,  the  plant  and  animal  body).     Thrive 


Generic  Characters  in  the  Coccaceae  205 

well  under  anaerobic  conditions.  Many  forms  fail  to  grow  on 
artificial  media;  none  produce  very  abundant  surface  growths. 
Planes  of  fission  often  parallel,  producing  pairs,  or  short  or  long 
chains,  never  packets.  Generally  stain  by  Gram.  Produce  acid 
in  dextrose  and  lactose  broth.     Pigment,  if  any,  white  or  orange. 

Genus  i,  Diplococcus  (Weichselbaum) :  Strict  parasites.  Not 
growing,  or  growing  very  poorly,  on  artificial  media.  Cells  nor- 
mally in  pairs  surrounded  by  a  capsule.  Includes  D.  pneumoniae 
Weich,  D.  W eichselhaumii  Trev.,  and  D.  gonorrheae  Neisscr. 

Genus  2.  Streptococcus  {V>\\\ro\.h.):  Parasites  (see  above).  Cells 
normally  in  short  or  long  chains  (under  unfavorable  cultural  condi- 
tions, sometimes  in  pairs  and  small  groups,  never  in  large  packets). 
Generally  stain  by  Gram.  On  agar  streak  effused,  translucent  growth, 
often  with  isolated  colonies.  In  stab  culture,  little  surface  growth. 
Sugars  fermented  with  formation  of  large  amount  of  acid.  Generally 
fail  to  liquefy  gelatin  or  reduce  nitrates.  Includes  S.  erysipelatos 
Fehleisen. 

Genus  3,  Aurococcus,  new  genus:  Parasites  (see  above).  Cells 
in  groups  and  short  chains,  very  rarely  in  packets.  Generally  stain 
by  Gram.  On  agar  streak  good  growth  of  orange  color.  Sugars 
fermented  with  formation  of  small  amount  of  acid.  Gelatin  often 
liquefied,  very  actively.  May  or  may  not  reduce  nitrates.  Includes 
A.  aureus  (Rosenbach). 

Genus  4,  AlhococcuSy  new  genus:  Parasites  (see  above).  Cells 
in  groups  and  short  chains  (never  in  packets).  Generally  stain  by 
Gram.  Growth  on  agar  streak  abundant  and  porcelain  white  in 
color.  Sugars  fermented  with  production  of  a  slight  amount  of 
acid.  Gelatin  hquefaction  and  nitrate  reduction  may  or  may  not 
occur.  Includes  A.  pyogenes  (Rosenbach),  A.  rhenanns  (Migula), 
A.  candicans  (Fliigge),  and  A.  canescens  (Migula). 

Subfamily  2,  Metacoccaceae  (W.  and  R.):  Facultative  parasites 
or  saprophytes.  Thrive  best  under  aerobic  conditions.  Grow  well 
on  artificial  media,  producing  abundant  surface  growths.  Planes  of 
fission  often  at  right  angles;  cell  aggregates  in  groups,  packets,  or 
zooglea  masses.  Generally  decolorize  by  Gram.  Pigment,  yellow  or 
red. 

Genus  5,  Micrococcus  (Hallier):  Facultative  parasites  or  sapro- 


2o6  C.-E.  A.  WiNSLow  AND  Anne  F.  Rogers 

phytes.  Cells  in  plates  or  irregular  masses  (never  in  long  chains 
or  packets).  Generally  decolorize  by  Gram.  Growth  on  agar 
abundant  with  formation  of  yellow  pigment.  Dextrose  broth  slightly 
acid,  lactose  broth  generally  neutral.  Gelatin  frequently  liquefied. 
Nitrates  may  or  may  not  be  reduced.  Includes  M.  orbicularis 
Ravenel,  M.  luteus  (Schroter)  Cohn,  and  M.  ochraceus  Rosenthal. 

Genus  6,  Sarcina  (Goodsir) :  Exactly  like  Micrococcus^  except 
that  division  occurs  under  favorable  conditions,  in  three  planes, 
producing  regular  packets.  Includes  S.  veniriculi  Goodsir,  S.  auran- 
tiaca  Fliigge,  S.  subflava  Ravenel,  S.  tetragena  (Mendoza)  Mig. 

Genus  7,  Rhodococcus,  new  genus:  Saprophytes.  Cells  in  groups 
or  regular  packets.  Generally  decolorize  by  Gram.  Growth  on 
agar  abundant,  with  formation  of  red  pigment.  Dextrose  broth 
slightly  acid,  lactose  broth  neutral.  Gelatin  rarely  liquefied.  Ni- 
trates generally  reduced  to  nitrites,  but  not  to  ammonia.  Includes 
R.  cinnabareus,  Fliigge,  R.  roseus  Fliigge,  R.  fulvus  Cohn,  R.  agilis 
(Ali  Cohen),  R.  rosaceus  Lindner,  and  R.  incarnalus  Gruber. 

V.    REFERENCES. 

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Buerger,  L.,  1904.     Med.  News,  85,  p.  1117. 

Chester,  F.  D.,  1901.     A  Manual  of  Determinative  Bacteriology,  New  York. 

Chester,  F.  D.,  1904.  Fifteenth  Annual  Report  of  the  Delaware  College  Agricul- 
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Clark,  H.  W.,  and  Gage,  S.  D.,  1905.     Eng.  News,  53,  p.   27. 

Committee  on  Standard  Methods  of  Water  Analysis,  1905.  Jour.  Infect. 
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Conn,  H.  W.,  1900.     Jour.  Boston  Soc.  Med.  Sciences,  4,  p.   170. 

Dunham,  E.  K.,  1903.     Abstr.,  Science,  N.   S.,   17,  p.   372. 

Ellis,  D.,  1902.     Centralbl.  f.   Bakt.,   Abt.   I,   Orig.,  33,   p.    i. 

Fischer,  H.,  1904.     Centralbl.  f.     Bakt.,  Abt.  I,  Orig.,  37,  p.  449. 

Flugge,    1896.     Die   Mikroorganismen,   Leipzig. 

Fuller,  G.  W.,  and  Johnson,  G.  A.,  1899.     Jour.  Exper.  Med.,  4,  p.  609. 

Gage,  S.  D.,  and  Phelps,  E.  B.,  1903.  Rep.  and  Papers  Amer.  Public  Health 
Assoc,  28,  1902  meeting,  p.  494. 

Galton,  F.,  1889.    (a)  Proc.  Roy.  Soc,  45,  p.  136;    (b)  Natural  Inheritance,  London. 

Howe,  F.,  1904.     Centralbl.  f.  Bakt.,  Abt.  I,  Orig.  36,  p.  484. 

Jordan,  E.  O.,  1903.     Jour.  Hyg.,  3,  p.  i. 

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Generic  Characters  in  the  Coccaceae  207 

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MiGULA,  W.,  1900.     System  der  Baklerien,  Band  II,  Jtna. 

Neumann,  R.,  1897.     Archiv  j.  Hyg.,  30,  p.  i. 

Otto,  R.,  1903.     Centralbl.  }.  Bakt.,  Abt.  I,  Orig.  34,  p.  44. 

Pearson,  K.,  1900.     The  Grammar  0}  Science,  London. 

Quetelet,  a.,  1846.  Letlres  ....  sur  la  theorie  des  probabilites,  appliquee  aux 
sciences  morales  et  politiques,  Brussels. 

Ripley,  W.  Z.,  1899.     The  Races  oj  Europe,   New  York. 

Robinson,  B.  L.,  1906.     Science,  N.  S.,  23,  p.  81. 

Smith,  T.,  1900.     Jour.  Boston  Soc.  Med.  Sciences,  4,  p.  95. 

Smith,  E.  F.,  1905.  Bacteria  in  Their  Relation  to  Plant  Diseases,  I,  Carnegie  Insti- 
tute,  Washington. 

StTLLiVAN,  M.  X.,  1905.     Jour.  Med.  Research,  14,  p.   109. 

Thorndike,  E.  L.,  1904.  Introduction  to  the  Theory  oj  Mental  and  Social  Measure- 
ments, New  York. 

Weston,  R.  S.,  and  Kendall,  A.  I.,  1902.  Rep.  and  Papers  Amer.  Public  Health 
Assoc,  27,  1901  meeting,  p.  402. 

Whipple,  G.  C,  1902.     Technol.  Quarterly,  15,  p.  127. 

WiNSLOw,  C.-E.  A.,  AND  Rogers,  Anne  F.,  1905.     Science,  N.  S.,   21,  p.  669. 

Woods,  F.  A.,  1906.     Mental  and  Moral  Heredity  in  Royalty,  New  York. 


THE     OCCURRENCE    OF    ORGANISMS     OF    SANITARY 
SIGNIFICANCE  ON  GRAINS  * 

Samuel  C.  Prescott, 

WITH  Co-operation  of 

Erastus   G.  Smith,  William  J.  Mixter  and   Selskar  M.   Gunn. 

In  the  development  of  bacteriology  as  applied  to  the  sanitary 
investigations  of  water  supply,  food  supply,  and  sewage  disposal, 
the  colon  bacillus  (B.  coli)  and  certain  streptococci  {Strept.  pyogenes) 
have  been  regarded  as  of  extraordinary  significance.  This  has 
been  especially  true  of  B.  coli,  which  has  been  studied  unceasingly 
and  by  a  large  number  of  investigators  almost  ever  since  its  dis- 
covery by  Emmerich  in  1885.  Having  been  early  shown  to  be  a 
constant  inhabitant  of  the  human  intestine,  and  present  there  in 
vast  numbers,  it  is  not  surprising  that  it  has  been  regarded  as  of 
great  sanitary  significance,  and  hence  one  of  the  most  important 
of  bacteria. 

The  streptococci  here  to  be  considered  have  received  less  atten- 
tion, as  they  were  more  recently  discovered,  but  as  they  have  been 
proved  to  be  present  in  sewage,  sewage  effluents,  and  polluted 
waters,  and  in  the  soil  which  receives  the  wastes  of  animal  life, 
these  too  have  been  regarded  as  characteristic  of  pollution,  and  their 
detection  has  served  as  a  striking  confirmation  of  the  evidence 
offered  by  the  finding  of  B.  coli,  as  an  index  of  fecal  contamination 
in  water. 

It  is  the  object  of  this  paper  to  present  a  record — incomplete, 
it  may  be — of  the  repeated  isolation  of  organisms  simulating  these 
"intestinal  forms"  in  habitats  other  than  those  named,  and  to  show 
that  they  are  actually  identical  in  character  with  B.  coli  and  Strept. 
pyogenes,  are  abundant,  and  of  constant  occurrence  on  the  surface 
of  grains,   and  in  products  of  milling. 

HISTORICAL  ACCOUNT. 

The  opinion  that  B.  coli  is  characteristic  of  pollution  from  human 
sources  only  was  long  since  proved  to  be  erroneous,  as  it  was  shown 
by    Dyar    and    Keith,'   Smith, ^   Flint, ^   Belitzer,^  and  Moore  and 

♦Received  for  publication  March  31,  1906. 

208 


Organisms  of  Sanitary  Significance  on  Grains        209 

Wright^  to  be  present  in  the  intestine  of  many  groups  of  animals. 
More  recently  a  number  of  investigators  in  PLurope  and  America 
have  reported  that  bacilli  in  all  respects  like  the  colon  bacillus  from 
the  human  intestine,  are  found  in  nature  where  there  is  no  evidence 
of  recent  or  direct  fecal  contamination.  Kruse*^  and  Weissenfeld^ 
declared  that  B.  coli  is  present  in  almost  all  v^^aters,  good  or  bad. 

In  1901  one  of  us^-^  isolated  from  grains  and  products  of  milling 
a  considerable  number  of  organisms  having  all  the  characteristics 
of  B.  coli,  and  Papasotiriu'°  obtained  the  same  results  in  Europe 
in  an  investigation  of  similar  scope.  Other  workers  have  also  pub- 
lished results  which  lead  to  the  conclusion  that  organisms  presenting 
the  characteristics  of  B.  coli  are  by  no  means  confined  to  the  intes- 
tinal tract  of  animals,  but  are  widely  distributed  in  nature. 

Investigations  on  the  occurrence  of  B.  coli  on  plants,  either 
healthy  or  diseased,  have  been  made  by  Gordan,"  Laurent,"  and 
Klein  and  Houston. '^  The  last-named  workers  examined  grains 
and  many  food  substances  for  B.  coli,  and  although  a  large  number 
of  samples  were  studied,  negative  results  were  obtained  in  most 
cases,  and  it  was  concluded  that  B.  coli  was  not  present  on  the  grains 
or  in  the  products  of  milling.  All  these  cultures  were  isolated  after 
a  prolonged  preliminary  cultivation,  generally  three  days  or  more, 
in  phenol  broth  or  phenol  gelatin — a  treatment  which  we  have  found 
generally  causes  the  colon  bacillus  to  be  killed  out  to  a  large  extent, 
if  continued  for  more  than  eight  hours,  and  which  therefore  may 
explain  in  part  their  negative  results. 

In  order  to  make  more  certain  that  the  organisms  found  on  grain 
were  not  merely  bacteria  having  some  of  the  more  striking  char- 
acteristics of  B.  coli,  we  undertook  in  1902  a  careful  comparison  of 
cultures  derived  from  the  human  intestine  and  from  grains.  The 
results  of  this  investigation  are  embodied  in  this  paper. 

In  the  autumn  and  winter  of  1904  one  of  us  (E.  G.  S.)'^  studied 
the  organisms  occurring  on  the  heads  of  grain  left  standing  in  fields 
far  removed  from  the  sources  of  pollution  with  fecal  matter,  and 
a  little  later  Metcalfe  conducted  an  investigation  of  the  flowers 
and  grains  from  rice-fields  in  South  Carolina,  but  where  evidences 
of  contamination  were  not  entirely  absent. 

The  investigation  of  the  occurrence  of  streptococci  was  lx*gun 


210  Samuel  C.  Prescott 

in  1894,  when  the  organisms  were  first  reported  by  Laws  and 
Andrewes.'^  Their  sanitary  importance  was  not  emphasized  until 
1899  and  1900,  when  Houston' ^  laid  special  stress  upon  the  fact 
that  streptococci  and  staphylococci  seem  to  be  characteristic  of  sewage 
and  animal  waste.  Horrocks'^  found  them  in  great  abundance 
in  sewage  and  in  polluted  waters,  and  showed  by  experiment  that 
they  outlived  the  colon  bacilli  in  samples  of  sewage,  a  subject  later 
explained'^  in  1902,  and  further  discussed  by  S.  K.  Baker  and  one 
of  the  writers'^"  the  following  year.  LeGros^'  in  a  monogroph  pub- 
lished in  1902  described  many  streptococci,  all  derived  from  the 
body  or  from  sewage. 

The  first  investigators  in  this  country  to  call  attention  to  these 
streptococci  were  Winslow  and  Miss  HunnewelP^  in  1901,  During 
the  same  year  they  were  reported  by  Gage^^  in  the  sewage  of  Lawrence. 

The  evidence  hitherto  presented  seems  to  show  the  streptococci 
to  be  associated  with  animal  bodies,  occurring  either  on  the  surface 
or  within  the  intestinal  tract.  The  work  here  described  tends  to 
show  that  these  organisms  are  found  in  abundance,  as  is  the  colon 
bacillus,  on  certain  substances,  at  least,  outside  the  body.  That 
they  have  not  been  earlier  reported  is  possibly  due  to  a  confusion 
of  these  organisms  and  certain  bacterium  forms,  as  is  suggested  by 
recent  work  by  Heinemann,^^  but  more  probably  because  no  system- 
atic search  for  them  has  been  made. 

The  methods  of  isolation  and  investigation,  and  the  comparison 
of  the  characteristics  of  the  organisms  derived  from  grain  with  those 
of  the  same  species  from  intestinal  sources,  may  now  be  most  con- 
veniently considered  separately. 

COMPARISON  OF   COLON   BACILLI   FROM   THE   DIFFERENT   SOURCES. 

Before  making  a  detailed  comparison  of  the  organisms  from 
the  two  sources,  it  is  necessary  to  define  the  characteristics  which 
we  have  regarded  as  typical  of  B.  coll.  These  may  be  expressed 
as  follows: 

Form. — Bacillus,  2-3  fi  long  by  0.5  m  wide,  with  rounded  ends. 

Grouping. — Occurs  singly,  or  in  short  chains  or  masses. 

Motility. — Actively  motile;  cilia  present. 

Spore   jormation. — None. 

Staining  reactions. — Stains  with  usual  reagents  and  by  Gram's  method. 


Organisms  of  Sanitary  Significance  on  Grains        211 

Gelatin  plate. — Thin,  small,  irregular  colonies — much  as  on  agar. 

Gelatin  stick. — Nail  growth;  small,  somewhat  spreading  colony  at  surface; 
gelatin  not  liquefied. 

Agar  plate. —Suriacc  colonies  opalescent,  nearly  circular,  edges  smooth.  Sul>- 
merged  colonies  clear  cut,  lenticular.  After  two  or  three  days  surface  colonies  take 
irregular  grape-leaf  forms. 

Agar  slope. — Twenty  hours:  lu.xuriant,  moist,  opalescent,  translucent,  white 
growth  narrowing  from   top  to  bottom. 

Nutrient  broth. — Twelve  hours:  distinct  turbidity;  i8  hours:  slight  sediment; 
after   two   days,    no   scum   on    surface. 

Litmus  milk. — Litmus  reddened  and  then  decolorized  in  from  12  to  18  hours; 
milk,   coagulated;  casein    not   rcdissolved. 

Potato. — Lu.xuriant,  dirty -yellowish  growth,  later  becoming  slightly  slimy;  potato 
not  discolored. 

Dextrose  broth. — Dextrose  fermented  in  from  6  to  12  hours  with  formation  of 

H        2 
acid  and  gas.     Gas  ratio  p^-  =-  ,     but  may  vary  even  with  the  same  stock. 

Saccharose  broth. — Fermented,  with  formation  of  acid  and  generally  some  gas! 

Lactose  broth. — Fermented;  acid  and  gas  formed;  with  less  gas,  but  gas  ratio 
more  constant  than  with  dextrose  broth. 

Maltose  broth. — Fermented,  with  formation  of  acid  and  gas;  similar  to  lactose. 

Litmus  lactose  agar  plate. — Litmus  reddened  in  less  than  24  hours;  bubbles 
of  gas  formed. 

Dunham's  solution. — Growth,  and  pronounced  nitroso-indol  reaction  in  three 
days. 

Anaerobic  agar  streak. — Thin,  transparent  growth,  somewhat  less  abundant  than 
in  aerobic  streak. 

Lactose  neutral  red  broth. — Gas  formed  and  color  generally  reduced  to  canar}'- 
yellow   (not   constant). 

METHODS   OF  ISOLATION   USED. 

In  the  earlier  experiments  cultures  of  the  bacteria  cxamineH 
were  obtained  by  making  infusions  of  the  grains  in  sterile  water 
and  plating  at  once  on  litmus  lactose  agar  without  any  prcliminar)' 
cultivation.  Variable  numbers  of  colonies  were  obtained,  some  of 
which  were  of  acid-producing  bacteria.  These  red  colonies  were 
then  "fished,"  the  masses  of  bacteria  shaken  up  in  sterile  water  and 
replated  on  litmus  lactose  agar.  From  the  typical  isolated  colonics 
on  this  plate  broth  cultures  were  made,  and  after  development 
these  were  used  as  stock  cultures  for  the  inoculation  of  other  media — 
viz.,  gelatin  stab,  litmus  milk,  agar  stroke,  dextrose  broth,  nitrate, 
and  Dunham  solution.  Hanging-drop  preparations  and  stained 
preparations  were  made  from  each  culture,  and  by  a  comparison 
of  the  reactions  and  morphological  features  with  those  of  B.  coli 


212  Samuel  C.  Prescott 

(intestinal)  it  was  found  that  25  out  of  a  total  of  47  cultures  isolated 
gave  typical  colon  characteristics,  while  several  more  failed  in  but 
a  single  test.     Nine  cultures  were  rejected. 

The  organisms  thus  obtained,  together  with  a  number  of  cultures 
of  supposed  B.  acidi  lactici  from  various  laboratories,  were  then 
subjected  to  actual  comparison,  side  by  side  with  22  cultures  of 
B.  coll  isolated  directly  from  feces,  or  from  waters  known  to  be 
sewage-polluted.  The  fecal  bacteria  were  obtained  by  preliminary 
cultivation  of  the  fecal  matter  in  dextrose  broth  for  four  to  eight 
hours,  and  then  plating  in  great  dilution  on  litmus  lactose  agar. 
It  was  found  that,  if  this  procedure  was  used,  a  nearly  pure  culture 
of  B.  colt  resulted;  but  if  the  preliminary  cultivation  was  prolonged, 
the  colon  bacilli  were  likely  to  be  overgrown  by  streptococci,  and 
frequently  were  entirely  lost. 

In  the  first  series,  cultures  from  the  sources  mentioned  below 
conform  to  the  following  characteristics:  Motile,  non-spore-forming 
bacilli  producing  turbidity  in  nutrient  broth,  characteristic  growth 
on  gelatin  plate,  surface  and  needle-growth  in  gelatin  stab;  growth 
on  potato  and  in  closed  arm  of  the  fermentation  tube;  grow  at 
body  temperature  and  are  facultative  anaerobes;  do  not  liquefy 
gelatin,  casein,  or  blood  serum;  produce  gas  in  dextrose,  lactose, 
and  saccharose  broth ;  nitrate  is  reduced,  indol  formed ;  milk  becomes 
acid  and  curdles. 

Five  cultures  of  B.  acidi  lactici  from  the  University  of  Chicago. 

Four       "      from  cornmeal. 

Six  "         "      buckwheat. 

Five        "         "      barley. 

Three     "         "      bran. 

One  culture  from  flour. 

One        "  "     breakfast  food. 

These  cultures  give  identical  results  and  can  in  no  way  be  differ- 
entiated from  the  18  cultures  mentioned  below,  which  are  of  undoubted 
intestinal  origin. 

Ten  cultures  from  feces. 


Two 

a 

the  Mystic  River. 

One 
One 
One 

''     Neponset  River, 
injected  peritoneum, 
pool  under  snow. 

One 
One 

One 

n 
n 
it 

driven  well  in  brewery. 

polluted    brook. 

meadow  at  Framingham. 

Organisms  of  Sanitary  Significance  on  Grains       213 

The  following  four  cultures  arc  without  doubt  "colon  forms," 
although  they  fail  to  give  typical  reactions  in  a  few  cases: 

Two   cultures   from   feces   failed   to   reduce   nitrates. 
One  culture  from  the  North  River,  Salem,  did  not  ferment  lactose. 
One  culture  from  feces  failed  to  ferment  any  of  the  three  sugars  and  rendered 
milk  alkaline. 

Thirteen  cultures  from  cornmeal,  corn,  milk,  flour,  malt,  buck- 
wheat, oats,  and  laboratory  stocks  of  B.  acidi  laclici  were  acid- 
producing  organisms  that  departed  widely  in  character  from  those 
just  described.  These  on  examination  proved  to  be  mostly  strep- 
tococci, although  during  this  portion  of  the  work  but  little  study 
was  given  to  them. 

fermenting  power. 

The  fermenting  power,   as  measured   by  acid   production,   was 

taken  as  a  further  means  of  comparing  these  apparently  identical 

organisms.     For  this   purpose   the   cultures   were  grown   in   2   per 

cent  dextrose  broth  at  37°  for  48  hours,  and  the  amount  of  acid 

N 
determined  by  titrating  5  c.c.  of  this   solution  against   —    NaOH. 

For  fairer  comparison  the  same  number  of  cultures  from  each  of 
the  two  sources  were  taken.     Twenty-one  cultures  of  B.  coli  from 

N 
unpolluted  sources  recjuired  an  average  of    1. 15  c.c.  of   —  NaOH 

to  neutralize  5  c.c.  of  the  cultures.     Twenty  cultures  of  B.  coli  from 

N 
feces  required  an  average  of  1. 13  c.c.  of  —  NaOH.     Ten  cultures 

N 
of   streptococci    required    an  average  of  1.35  c.c.  of  —  NaOH. 

PATHOGENIC   PROPERTIES. 

As  a  final  test  for  this  series  the  pathogenic  power  of  the  bacteria 
toward  guinea-pigs  was  studied.  Three  cultures  were  chosen  at 
random  from  each  of  the  two  lots,  and  fresh  broth  cultures  prepared 
for  use  as  an  inoculating  medium. 

For  the  first  experiment  0.5  c.c.  of  a  culture  from  cornmeal 
was  injected  subcutaneously  into  a  healthy  guinea-pig,  and  a  like 
amount  of  a  culture  from  an  infected  peritoneum  into  another  animal 
of  equal  weight.  The  animals  exhibited  symptoms  of  fever  after 
about  48  hours,  but  on  the  third  day  appeared  more  nearly  normal. 


214  Samuel  C.  Prescott 

On  being  autopsied,  each  showed  a  mass  of  inflamed  tissue  at  the 
point  of  inoculation,  and  from  this  inflamed  portion  the  bacilli 
were  recovered  in  large  numbers. 

As  a  second  test  two  guinea-pigs  were  inoculated  subcutane- 
ously  with  i  c.c.  of  the  same  cultures  as  were  used  in  the  previous 
experiment.  In  this  experiment  the  animals  appeared  dull,  and 
their  temperature  fell  slightly  in  six  hours,  while  at  the  end  of  24 
hours  the  temperature  had  risen  above  normal,  and  the  animals 
were  decidedly  sick  and  weak,  and  showed  swelling  at  the  point  of 
inoculation.  At  the  end  of  48  hours  the  temperature  had  still  further 
risen,  but  no  further  change  was  noticeable.  One  of  these  animals 
was  autopsied,  the  other  was  kept,  and  ultimately  fully  recovered. 

A  third  pair  of  animals  was  inoculated  intraperitoneally  with 
I  c.c.  of  cultures  of  B.  acidi  lactici  and  a  culture  from  feces  respec- 
tively. In  this  case  the  animals  showed  much  stronger  lesions, 
although  death  was  not  caused  in  48  hours.  As  in  the  previously 
described  experiments,  no  difference  could  be  observed  in  the  beha- 
vior of  the  cultures  from  the  two  sources.  On  chloroforming  the 
animals,  cultures  taken  from  peritoneum  and  heart's  blood  showed 
the  presence  of  pure  cultures  of  the  germs  used  for  inoculation. 

Two  more  animals  were  infected  intraperitoneally,  one  with 
another  culture  of  B.  acidi  lactici,  and  the  other  with  another  culture 
from  feces.  In  this  experiment  the  amount  of  the  dose  was  1.5  c.c. 
in  each  case.  A  sharp  temperature  drop  was  observed  in  six  hours, 
and  in  24  hours  each  of  the  animals  was  dead.  Cultures  made 
from  blood  and  peritoneum  gave  a  pure  growth  of  B.  coli. 

These  experiments  show  conclusively  that  the  pathogenic  power 
of  the  organisms  derived  from  grain  is  fully  as  great  as  with  the 
intestinal  or  more  nearly  parasitic  colon  bacilli,  and,  we  believe, 
offers  a  strong  support  to  the  proof  of  their  actual  identity.  Some 
more  recent  work  by  Gordan^-*  has  shown  the  pathogenic  properties 
for  white  mice  of  organisms  derived  from  bran. 

In  all  investigations  thus  far  reported  some  doubt  might  be  cast 
on  the  integrity  of  the  samples,  or  at  least  there  is  a  possibility  of 
contamination  from  handling  or  manufacture.  To  eliminate  this 
objection,  the  following  work  was  carried  out  during  the  late  autumn 
and  winter  of  1904.     In  November  a  field  of  rye  was  found  by  one 


Organisms  of  Sanitary  Significance  on  Grains        215 

of  us  in  western  Massachusetts,  which,  owing  to  the  scanty  growth, 
had  not  been  cut.  The  field  is  of  light  soil,  on  a  sandy,  level,  open 
plain,  and  situated  well  back  from  a  little-traveled  country  road, 
and  far  from  human  habitation.  Inquiry  showed  that  the  field 
had  not  been  fertilized, and  that  no  cattle  had  ranged  through  the 
grain  during  the  year.  This  stand  of  grain,  therefore,  may  be 
taken  as  a  typical  open-country  growth,  free  from  contaminating 
influences.  From  this  field  heads  of  grain  were  picked  with  steri- 
lized forceps  and  put  into  sterilized  glass  tubes.  These  heads  were 
incubated  separately  in  broth  for  24  hours,  and  then  cultures  on 
litmus  lactose  agar  were  prepared,  following  the  usual  procedure. 
On  December  10  and  January  17  two  more  lots  of  grain  heads  were 
treated  in  similar  manner.  In  all,  34  heads  from  this  field  of  rye 
were  studied,  and  from  six  of  them  organisms  were  isolated  giving 
the  reactions  of  the  colon  bacillus  on  all  ordinary  media  and  with 
neutral  red  lactose  broth.  It  will  be  recalled  that  these  heads  were 
taken  at  random  over  the  whole  field  after  they  had  stood  through 
the  storms  of  the  fall  and  snows  of  the  early  winter.  Other  heads 
of  rye  gave  acid-producing  organisms,  but  not  those  exhibiting  the 
typical  colon  reactions  in  all  respects. 

The  occurrence  of  acid-  and  gas-producing  organisms  under  these 
conditions  has  been  known  for  several  years,  as  it  was  shown  by 
Underwood  and  one  of  us^^  in  1897,  ^^d  again  in  1898,  that  acid- 
producing  organisms  of  numerous  types  are  of  common  occurrence 
upon  the  ears  of  sweet  corn  beneath  the  husks,  and  upon  the  surfaces 
of  peas  in  the  pod,  with  practically  complete  protection  from  con- 
tamination from  the  external  world. 

Although  in  the  investigations  cited  only  the  resistant  types  of 
organisms  were  thoroughly  studied,  the  more  recent  work  shows 
the  strong  probability  that  organisms  of  the  colon  and  Strcpt.  pyogenes 
types  were  likewise  present.  Further  evidence  on  this  point  is  afforded 
by  some  experiments  begun  by  us  in  Wisconsin,  in  which  we  exam- 
ined a  large  number  of  kernels  of  corn  from  ears  carefully  selected 
in  the  field  because  of  their  isolation  from  polluting  substances  and 
further  protection  from  closely  packed  husks.  Although  the  cxjicri- 
ments  were  not  carried  out  in  the  great  detail  of  those  previously 
described,  the  presumptive  tests  first  employed  gave  strong  evidence 
of  the  abundance  of  these  bacteria  on  the  grain. 


2i6  Samuel  C.  Prescott 

EXAMINATION   OF  GRAINS   FOR   STREPTOCOCCUS   PYOGENES, 

The  investigations  hitherto  described  have  aimed  to  point  out 
the  common  occurrence  of  B.  coli  on  the  Gramineae. 

To  give  greater  interest  and  value  to  this  work,  it  was  deemed 
desirable  to  extend  its  scope  by  an  inquiry  into  the  character  and 
constancy  of  occurrence  of  streptococci. 

To  put  the  problem  in  more  concrete  form:  Are  the  two  classes 
of  organisms,  colon  bacilli  and  streptococci,  of  constant  occurrence, 
and  if  so,  do  they  present  any  biological  relation  to  each  other  simi- 
lar to  that  existing  in  sewage  or  polluted  water  ? 

ISOLATION  OF  BOTH  B.    COLI   AND  STREPTOCOCCUS   pyogenes. 

It  has  been  shown  by  one  of  us  that  when  B.  coli  and  Strept. 
pyogenes  are  both  present  in  a  sample  of  water  or  sewage,  inocu- 
.lation  from  the  sample  into  dextrose  broth  and  incubation  at  37° 
gives  a  development  of  B.  coli  in  the  first  few  (six  to  twelve)  hours, 
while  at  the  end  of  36  to  48  hours  the  streptococci  are  predominant. 
Applying  this  procedure  to  grain,  we  have  found  that  of  a  very 
large  number  of  experiments  nearly  all  have  shown  the  occurrence 
of  both  organisms. 

Thirteen  cultures  of  the  colon  bacillus  isolated  from  the  following 
sources  proved  to  be  identical: 

Seven  cultures  from  wheat. 
Two  "  "     buckwheat. 

Two  "  "      rye. 

One    cuhure    from    barley. 
One  "  "       oats. 

The  purpose  of  the  colon  isolations  was  to  confirm  earlier  results 
and  determine  if  both  kinds  develop  true  to  the  type. 

Plating  from  the  dextrose  broth  culture  on  litmus  lactose  agar 
as  soon  as  gas  formation  was  well  begun  gave  a  predominance  of 
the  colon-type  organisms,  while  after  24  hours  the  streptococci 
were  present  in  abundance.  We  have,  therefore,  not  only  constant 
occurrence  of  both  types,  but  the  same  course  of  development  in 
dextrose  broth  cultures  with  grains  and  with  sewage — a  fact  which 
may  have  great  practical  significance  in  sanitary  work. 

Comparison  of  this  last  series  of  cultures  with  the  first  series  will 
show  the  constancy  in  biochemical  reactions  and  morphological 
features  of  the  colon  bacilli  from  all  sources. 


Organisms  of  Sanitary  Significance  on  Grains        217 

This  set  of  cultures  of  B.  coli  was  further  compared  with  the 
intestinal  organisms  by  a  study  of  the  staining  relations,  with  the 
result  that  the  organisms  were  found  to  react  toward  dyes  in  the  same 
manner,  with  the  usual  staining  methods  as  well  as  by  Gram's  method. 

COMPARISON   OF   STREPTOCOCCUS  PYOGENES    FROM   GRAIN 
AND   FROM   INTESTINAL   SOURCES. 

Having  shown  the  constant  occurrence  of  streptococci,  their  com- 
parison with  intestinal  organisms  of  the  same  genus  is  of  interest. 

The  form  of  most  significance  in  sanitary  work  is  Slrepl.  pyogenes, 
the  "sewage  streptococcus."  This  organism  presents  the  following 
characteristics : 

Form. — Coccus,    im  in   diameter. 

Grouping. — Occurs  in  short  chains,  often  in  pairs. 

Motility. — Non-motile. 

Spore  formation. — None. 

Gelatin  plate. — Small  colonies,  similar  to  those  on  agar;  no  liquefaction. 

Gelatin  stab. — Nail  growth,  apparently  made  up  of  isolated  colonies;  very  slight 
spreading   on    surface. 

Agar  plate. — Colonies  small;  under  low  power  somewhat  irregular  in  form; 
edges  smooth. 

Agar  streak. — Faint  dotted  growth  in  24  hours. 

Broth. — Faint  turbidity  and  perceptible  sediment  in  18  hours;  on  shaking,  sedi- 
ment rises  in  spiral. 

Litmus  milk. — Twelve  hours:  slightly  acid;  litmus  slightly  decolorized;  18  hours, 
strongly  acid;  36  hours,  coagulated. 

Potato. — Invisible  or  hardly  perceptible  white  growth  after  three  days. 

Dextrose  broth. — Eighteen  hours:  strongly  acid;  no  gas;  sediment  and  slight 
turbidity  in  both  arms. 

Saccharose  broth. — Eighteen  hours:  sediment  and  turbidity,  but  no  evidence 
of  change  of  sugar. 

Lactose   broth. — Same   as   dextrose   broth. 

Maltose  broth. — Same   as  dextrose  broth. 

Litmus  lactose  agar  plate. — Twelve  hours:  litmus  reddened;  colonies  small 
with  pink  tint  as  if  colored  by  litmus. 

Dunham's   solution. — Apparently   no   growth;  no   indol    produced. 

Anaerobic  agar  streak. — Dotted  growth  similar  to  aerobic,  but  rather  less  strong. 

The  cultures  isolated  from  the  grains  were  compared  side  by  side 
with  a  set  of  streptococci  isolated  directly  from  feces.  Only  those 
organisms  showing  typical  morphological  appearance  in  stained  prepa- 
ration were  used  in  this  comparison.  Three  cultures  of  Strept.  pyo- 
genes, eight  from  rye,  six  from  oats,  three  from  buckwheat,  one  from 
wheat,  and  eight  cultures  from  feces  all  proved  to  be  alike  in  their  cul- 


2i8  Samuel  C.  Prescott 

tural  features  as  well  as  morphologically.  They  likewise  exhibit  the 
same  staining  reactions,  colorizing  readily  with  the  usual  stains  and 
also  by  Gram's  method. 

COMPARISON  OF   ACID   PRODUCTION. 

The  comparison  of  acid-producing  power  of  the  organisms  was 
made  as  a  further  test  of  their  identity.  For  this  purpose  we  made  use 
of  I  per  cent  lactose  broth  and  i  per  cent  maltose  broth,  both  made  with 
reaction  o.o  at  the  beginning  of  the  experiment. 

.       .  .        N 

The  acidity  was  determined  by  titration  against  —  NaOH,  using 

phenolphthalein  as  an  indicator. 

The  acid  in  the  lactose  broth  cultures  was  measured  after  96  hours, 
and  in  the  maltose  broth  cultures  after  48  hours. 

Five  cultures  of  streptococci  from  grains  required  an  average  of 

N 
2.48  c.c.  of  —  NaOH  to  neutralize  =:  c.c.  of  the  cultures  in  lactose 
20  -^ 

broth. 

Five  cultures  from  feces   required   an   average   of   2.51    c.c.    of 

N 

—  NaOH  cultures  in  lactose  broth. 
20 

Six  cultures  of  streptococci  from  grains  required  an  average  of 

N 
1 .  78  c.c.  of  —  NaOH  to  neutralize  k  c.c.  of  cultures  in  maltose  broth. 

Five  cultures  from   feces  required  an  average   of  1.83    c.c.    of 

N 

—  NaOH  cultures  in  maltose  broth. 
20 

These  results  show  that  there  is  no  essential  difference  in  the  acid- 
producing  power  of  the  organisms.  Taking  the  averages — 2 .  48,  2  . 5 1 , 
1.78,  and  1.83,  respectively — it  is  obvious  that  the  differences  are 
quite  within  the  experimental  error  in  a  determination  by  this  method. 

Judged  by  biochemical  or  microscopical  characters  and  fer- 
menting powers,  it  is  impossible  to  distinguish  between  the  organisms 
from  the  two  sources,  and  we  must  regard  the  species  Strept.  pyogenes, 
as  well  as  B.  coli,  as  having  a  very  wide  distribution  in  nature,  and 
not  merely  associated  with  animal  organisms.  Its  occurrence  appears 
to  us  to  be  in  some  way  correlated  with  the  presence  of  carbohy- 
drate food,  and  it  is  evident  that  it  can  obtain  this  either  in  the  intes- 


Organisms  of  Sanitary  Significance  on  Grains        219 

tine  of  man  or  on  the  developing  flower  or  seed  of  a  plant,  especially 
one  in  which  sugar  storage  takes  place  abundantly  as  in  the  grains. 

summary  and  conclusions. 

As  a  result  of  the  comparative  investigations  which  have  just 
been  described,  we  have  succeeded  in  establishing  a  specific  agree- 
ment between  the  organisms  corresponding  to  the  graminal  B.  colt 
and  Strept.  pyogenes  and  the  intestinal  B.  coll  and  Strept.  pyogenes 
in  the  following  ways:  (i)  in  the  cultural  reactions;  (2)  in  the 
morphological  and  biological  characteristics;  (3)  in  the  fermen- 
tative powers  and  for  the  colon-like  forms;  (4)  in  the  pathogenic 
properties. 

The  evidence  is  so  positive  and  so  complete  as  to  lead  to  the  con- 
clusion that  the  identity  of  these  so-called  groups  is  absolute. 

It  would  seem  as  if  the  study  of  the  distribution  of  these  germs 
had  hitherto  been  neglected  in  much  the  same  way  as  was,  until 
recently,  the  case  with  the  tetanus  bacillus  as  well,  with  one  marked 
difference.  The  latter  has  always  been  thought  to  be  an  inhabitant 
of  garden  earth,  and  out  of  its  normal  environment  when  in  the 
human  body.  Now  it  has  been  found  to  be  always  present  in  the 
fecal  discharges  of  many  ruminants,*"^  and  we  come  to  the  question: 
Which  is  the  normal  environment — the  earth,  the  animal  body, 
or  both  ?  The  colon  bacillus,  on  the  contrary,  has  always  been 
considered  the  typical  intestinal  bacillus,  and  abnormal  elsewhere. 
Our  work  has  led  us  to  suppose  that  it  is  normally  present  either 
in  or  on  many  vegetable  tissues,  and  we  are  inclined  to  believe  that 
investigators  who  have  reported  B.  colt  in  vegetable  tissues  have 
not  necessarily  found  germs  of  immediate  intestinal  origin,  as  gen- 
erally suggested,  but  simply  were  not  aware  of  its  wide  distribution. 

The  results  obtained  in  this  investigation  possess  considerable 
interest  from  both  the  theoretical  and  the  practical  standpoints. 
Naturally,  question  first  arises  as  to  their  origin.  Have  these  organ- 
isms been  transported  through  the  air  as  dust,  or  carried  by  insects 
contaminated  with  animal  excrement,  and  thus  gained  access  to 
the  grain  ?  Assuming  this  to  be  the  case,  it  is  dilTicult  to  explain 
the  numbers  and  persistence  of  these  forms  on  grain  far  removed 
from  contaminating  materials  in  unfertilized  fields.     \\  hile  admit- 


220  Samuel  C.  Prescott 

ting  the  possibility  of  this  view,  it  seems  to  us  unlikely.  The  other 
alternative  is  that  these  organisms  are  constantly  associated  with, 
and  of  normal  occurrence  upon,  the  grain  heads;  that  they  find  suf- 
ficient sustenance  to  support  life,  or  even  to  increase  in  numbers; 
in  other  words,  that  they  show  a  mild  form  of  association  or  semi- 
parasitic  relation  with  the  plants  on  which  they  develop.  It  seems 
.unreasonable  that  organisms  exhibiting  such  marked  identity  in  all 
details  of  growth  and  cultural  behavior  should  be  regarded  as  of 
different  species.  More  likely  these  organisms  bear  much  the  same 
relation  to  the  sugary  heads  that  B.  suhtilis  does  to  the  stalks  of  hay, 
or  Strept.  hollandicus  to  Pinguicula,  or  the  nitrogen- fixing  bacteria  to 
the  legumes.  With  this  view,  it  is  easy  to  see  how  the  organisms 
could  find  their  way  to  the  animal  intestine  where  the  temperature 
and  food  conditions  for  rapid  development  are  ideal.  Nor  is  it  sur- 
prising, in  view  of  the  careful  and  exhaustive  search  for  bacterial 
proofs  of  contamination,  that  the  organisms  in  the  intestine  should 
first  be  sought,  and,  because  of  their  abundance,  regarded  as  origi- 
nating in  this  habitat.  The  explanation  made  possible  by  regarding 
these  species  of  bacteria  as  constantly  associated  with  the  starch-pro- 
ducing grains  is  not  only  simpler,  but  in  our  opinion  fits  in  more 
readily  with  all  the  observed  facts.  Probably  many  more  such  asso- 
ciations of  certain  species  of  bacteria  with  plants  which  can  supply 
their  food  requirements  will  be  observed  in  the  future,  and  here  is 
certainly  an  interesting  field  for  research. 

While  thus  of  interest  from  the  general  biological  standpoint, 
it  is  in  their  bearing  on  the  questions  relating  to  sanitary  bacterio- 
logical practice  that  our  observations  have  the  utmost  importance. 
Whatever  may  be  the  origin  of  the  individuals  whence  these  organisms 
have  been  developed,  their  presence  on  the  grains  suggests  sources 
of  the  colon  bacilli  and  streptococci  in  water  other  than  direct  sewage 
pollution.  Whether  the  organisms  find  their  way  from  grain  to 
natural  waters  can  be  determined  only  by  an  exhaustive  study  of 
the  bacteriology  of  unpolluted  streams  in  a  new  grain-producing 
region  such  as  western  Canada. 

Certainly  the  presence  of  small  numbers  of  these  organisms 
must  be  interpreted  with  the  utmost  discretion.  As  a  result  of 
careful  study,  we  are  not  inclined  to  believe  that  our  results  invali- 


Organisms  of  Sanitary  Significance  on  Grains        221 

date  the  bacteriological  examination  of  waters,  for  the  eastern  states 
at  least,  except  in  the  immediate  neighborhood  of  grist-mills,  etc.; 
but  they  certainly  render  it  imperative  that  great  care  should  always 
be  used  in  the  interpretation  of  results.  Certainly  the  old  and  fast 
rule  concerning  the  significance  of  the  presence  of  any  "intestinal 
forms"  as  prima  facie  evidence  of  sewage  pollution  must  be  most 
discriminately  applied. 

Whether  our  results  will  involve  any  change  in  the  criteria  now 
in  vogue  in  judging  of  the  character  of  a  water  can  only  be  a  matter 
of  conjecture  until  work  on  the  waters  of  grain-producing  regions 
has  been  carefully  conducted.  It  seems  obvious  that  at  least  inspec- 
tion of  watersheds  by  the  trained  bacteriologist  is  necessary,  and 
that  no  adverse  opinion  should  be  based  upon  the  results  of  a  single 
bacteriological  examination  without  eliminating  this  possible  source 
of  entrance  of  bacteria  of  supposedly  suspicious  character. 

REFERENCES. 

1.  Dyar,  H.  G.,  and  Keith,  S.  C,  Jr.     Tech.  Quart.,  1893,  6,  p.  256. 

2.  Smith,  T.     Centralbl.  f.  Bakt.  1895,  17,  p.  726. 

3.  Flint,  J.  M.     Jour.  Am.  Med.  Assoc,  1896,  26,  p.  410. 

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6.  Kruse,  W.     Ztschr.  j.  Hyg.,  1894,  17,  p.  i. 

7.  Weissenfeld,  J.     Ibid.,  1900,  35,  p.  76. 

8.  Prescott,  S.  C.     Science,  1902,  15,  p.  363. 

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10.  Papasotiriu,  J.     Archiv.  j.  Hyg.,  1902,  41,  p.  204. 

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12.  Laurent,  Emile.     Ann.  de  I'lnst.  Pasteur,  1899,  13. 

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15.  Metcalf,  H.      Science,  1905,  N.  S.  22,  No.  562,  pp.  439-41. 

16.  Laws  and  Andrewes.     Report  to  the  London  County  Council,   1894,  No.   216. 

17.  Houston,  A.  C.     Ann.  Rep.  Local  Gov.  Board,  containing  Rep.  of  Med.  Off., 
1899;  ibid.,  1900. 

18.  HORROCKS,   W.   H.     Bacteriological  Examination  of   Water.   London,    1901. 

19.  Prescott,  S.  C.     Science,  1902,  N.  S.,  16,  pp.  408,  671. 

20.  Prescott,   S.   C,  and  Baker,  S.  K.     Jour.  Infect.  Dis.,  1904,  i,  p.  193;  Public 
Health,  1904,  p.  29.     Report  for  1903,  pp.  369-85. 

21.  LeGros,  F.-L.     Monographie  des  streptocoques  et  des  agents  des  septicemics  mcta- 
diphtheriqms,   particulihement  des  diplocoques,   Paris,    1902. 


222  Samuel  C.  Prescott 

22.  WiNSLOW,  C.-E.  A.,  AND  HtJNNEWELL,  Miss  M.  P.     Jour.  Med.  Res.,  1902,  3, 
N.  S.,  p.  502. 

23.  Gage,  S.  DeM.     3jrd  Ann.  Rep.  Mass.  State  Board  of  Health,  1901. 

24.  GORDAN.     Die  landwirthsch.  Versuchsstation,  60,  p.  91. 

25.  Prescott,  S.  C.,  and  Underwood,  W.  L.     Tech.  Quart.  1898,  11,  pp.  6-30. 

26.  Medicine,    April,    1902,    p.    313. 

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A  STUDY  OF  THE  NUMBERS  OF  BACTERIA  DEVELOP- 
ING AT  DIFFERENT  TEMPERATURES  AND  OF  THE 
RATIOS  BETWEEN  SUCH  NUMBERS  WITH  REF- 
ERENCE TO   THEIR  SIGNIFICANCE  IN  THE 
INTERPRETATION  OF  WATER 
ANALYSIS.* 

Stephen  DeM.  Gage. 

In  the  judgment  of  the  quality  of  a  water  many  factors  must  be 
taken  into  consideration  by  the  person  making  the  interpretation. 
Until  within  a  few  years  it  was  the  custom  to  base  such  an  interpreta- 
tion upon  the  sanitary  survey  of  the  source  of  the  water  and  upon  the 
results  of  a  few  chemical  determinations.  As  the  value  of  sanitary' 
analysis  became  better  known  and  more  analyses  were  made,  various 
discrepancies  were  noted  between  the  different  factors  used  in  the 
interpretation;  and  the  correct  interpretation  became  more  compli- 
cated as  the  number  of  chemical  determinations  was  increased. 
With  the  inception  of  bacteriological  methods  of  analysis,  it  was 
beheved  that  a  determination  of  the  number  of  bacteria  would  prove 
a  good  criterion  to  the  character  of  a  water.  Extended  examinations, 
however,  proved  otherwise,  and  the  determination  of  numbers  of  bac- 
teria became  merely  an  additional  factor  to  be  used  in  the  interpre- 
tation. More  recently  determinations  of  specific-groups  of  bacteria, 
such  as  B.  colt,  the  sewage  streptococcus,  and  the  B.  sporogenes  groups, 
have  been  largely  exploited,  and  have  proved  of  more  or  less  value  in 
indicating  the  character  of  the  water;  but  as  the  distribution  of  these 
groups  has  become  better  understood,  the  results  of  these  specific 
bacterial  tests  have  also  been  found  to  require  interpretation.  As  the 
subject  stands  today,  the  sanitary  quality  of  a  water  is  usually  deter- 
mined by  a  critical  study  of  the  data  obtained,  first,  from  a  sanitary 
survey  of  the  source  of  the  water;  second,  from  the  results  of  a  num- 
ber of  chemical  procedures;  and,  third,  from  the  results  of  bacterio- 
logical tests.  The  chemical  procedures  of  most  value  arc  determina- 
tions of  the  nitrogen  as  free  ammonia,  as  albuminoid  ammonia,  as 

*Received  for  publication  February  13,  1906. 

223 


224  Stephen  DeM.  Gage 

nitrates,  and  as  nitrites;  of  the  chlorine  and  of  the  oxygen  consumed 
from  permanganate ;  although  determinations  of  odor,  color,  turbidity, 
iron,  hardness,  etc.,  may  enter  into  the  consideration  of  the  use  of  a 
water  for  one  purpose  or  another.  In  the  bacterial  analysis  we  have 
the  determination  of  the  numbers  of  bacteria,  usually  determination 
of  the  presence  or  absence  of  bacteria  of  the  colon  type,  and  occa- 
sionally determinations  of  bacteria  of  the  sporogenes  or  sewage 
streptococcus  type.  The  analyst  having  in  his  possession  the  above 
data  must  know  the  significance  of  high  or  low  values  of  each  indi- 
cating factor.  Even  with  the  number  of  factors  at  hand,  many  of 
the  data  are  often  contradictory,  and  a  correct  interpretation  of  the 
quality  of  the  water  is  not  always  possible. 

The  weakness  in  a  sanitary  survey  is  that,  while  it  may  show  pollu- 
tion, it  does  not  always  show  whether  that  pollution  is  serious, 
or  whether  the  water  has  become  purified;  and,  furthermore,  it  may 
not  reveal  sources  of  pollution  which  can  often  be  detected  by  analytical 
means.  The  chemical  examination  can  show  us,  when  we  know  the 
character  of  the  source  of  the  water,  whether  the  water  has  or  has  not 
been  polluted,  and  the  extent  of  the  pollution.  It  cannot,  however, 
always  reveal  whether  the  water  in  its  present  state  is  safe  or  danger- 
ous for  domestic  use ;  and  this  remains  for  the  bacteriologist  to  deter- 
mine. The  sanitary  survey  and  the  chemical  analysis  of  water  have 
been  sufficiently  studied,  so  that  our  knowledge  of  the  causes  of  varia- 
tions and  fluctuations  in  the  various  factors  is  well  grounded.  From 
the  side  of  the  bacterial  examination,  however,  much  remains  to  be 
investigated.  We  have  a  good  working  knowledge  of  the  significance 
and  fluctuation  in  the  numbers  of  bacteria  in  different  waters  when 
determined  by  gelatin  or  agar  plates  after  incubation  at  a  tempera- 
ture of  20°  C.  We  are  rapidly  acquiring  a  good  working  knowledge 
of  the  true  significance  of  the  appearance  of  B.  coli,  the  sewage  strep- 
tococcus, and  B.  sporogenes  in  water  of  various  classes.  Many  bac- 
teriological factors,  however,  which  may  be  of  value  have  received 
little  or  no  attention. 

It  is  the  purpose  of  the  writer  in  the  present  paper  to  introduce  data 
bearing  on  the  numbers  of  bacteria  which  develop  on  plates  incubated 
at  different  temperatures,  and  the  ratios  between  such  numbers,  for 
different  classes  of  waters,  in  an  endeavor  to  show  that  factors  so 


Bacteria  Developing  at  Different  Temperatures     225 

obtained  may  have  an  important  i)lace  in  judging  the  character  of  a 
water  by  analytical  means.  In  a  large  number  of  laboratories,  where 
the  routine  work  consists  in  the  control  of  the  water  supplies  of  large 
cities  or  in  the  control  of  water  filters,  almost  complete  reliance  is 
placed  on  the  results  of  bacteriological  examinations  in  judging 
whether  such  waters  are  of  the  required  (quality.  In  such  cases  any 
additional  factors  which  will  assist  in  a  more  accurate  judgment,  or 
any  change  in  the  procedure  which  will  enable  such  a  judgment  to  be 
arrived  at  more  quickly  than  under  present  conditions,  would  be  of 
inestimable  value  in  protecting  the  consumers  from  the  effects  of  any 
sudden  change  in  the  quality  of  such  waters. 

Media  for  bacterial  counts. — It  is  well  known  that  gelatin  and  agar 
do  not  show  us  the  total  bacterial  content  of  a  water,  much  higher 
numbers  being  obtained  when  other  and  differently  constituted  media 
are  employed;  and,  furthermore,  slight  changes  in  the  composition 
of  the  culture  media  may  cause  considerable  variations  in  the  num- 
bers of  bacteria  developing  on  those  media.  These  points  have  been 
very  thoroughly  investigated  by  Fuller,'  Hesse  and  Nieder,^  Whipple,' 
G.  Hesse,'*  the  writer,^  and  others,  and  their  consideration  need  not 
be  entered  upon  at  this  time.  Gelatin  and  agar  continue  to  be  the 
media  most  commonly  employed  in  quantitative  bacterial  determina- 
tions, for  several  reasons,  the  principal  ones  being  that  the  interpreta- 
tion obtained  by  their  use  is  well  grounded,  and  that  with  the  newer 
media  the  time  required  to  make  a  bacterial  examination  is  lengthened 
instead  of  shortened.  It  is  largely  true,  moreover,  that  while  gelatin 
and  agar  do  not  show  us  the  total  bacterial  content  of  a  water,  we  are 
able  by  their  use  to  obtain  a  knowledge  of  a  fairly  representative  sec- 
tion of  that  bacterial  content.  The  practice  as  to  the  use  of  gelatin 
or  agar  varies  widely  in  different  laboratories.  The  principal  advan- 
tage obtained  in  the  use  of  gelatin  instead  of  agar  is  that  a  determina- 
tion of  the  number  of  liquefying  bacteria  is  possible — a  factor  of  little 
practical  value,  except  when  dealing  with  sewage  and  the  effluents 
from  sewage  disposal  works,  and  which  is  more  than  offset  by  the 
greater  ease  with  which  agar  plates  may  be  manipulated. 

During  more  than  15  years  it  has  been  the  custom  to  employ 
agar  in  routine  work  at  Lawrence,  the  Lawrence  agar  being  identical 
in  composition  with  that  recommended  by  the  Committee  on  Standard 


226  Stephen  DeM.  Gage 

Methods  of  Water  Analysis,*^  except  that  it  contains  only  i  per  cent  of 
agar,  instead  of  i .  5  per  cent  as  recommended  by  that  committee. 
Litmus-lactose  agar  as  a  substitute  for  gelatin  or  agar  in  determining 
the  numbers  of  bacteria  is  slowly  gaining  a  foothold  in  many  labora- 
tories, its  advantage  being  that  it  permits  a  distinction  to  be  made 
between  the  types  of  bacteria  which  do  and  do  not  produce  acid  fer- 
mentation of  lactose,  the  determinations  of  the  presence  and  numbers 
of  the  fermenting  organisms  being  of  considerable  significance,  as  will 
be  shown  further  on. 

Temperature  0}  incubation. — The  determination  of  numbers  of 
bacteria  on  plates  which  have  been  incubated  at  20°  C.  or  at  room 
temperature  is  the  usual  practice.  In  many  laboratories,  where  gela- 
tin is  employed  as  the  routine  medium,  the  plates  are  incubated  at  a 
somewhat  lower  temperature,  usually  about  15°  C,  in  order  to  prevent 
the  rapid  growth  of  the  liquefying  bacteria  and  to  minimize  the  errors 
due  to  such  liquefaction.  In  other  laboratories,  where  agar  is 
employed,  it  is  the  custom  to  incubate  the  plates  at  temperatures  of 
23°  to  26°  C,  in  the  endeavor  to  obtain  higher  counts  with  shorter 
periods  of  incubation.  The  recommendation  of  the  Committee  on 
Standard  Methods,  that  a  uniform  temperature  of  20°  C.  be  employed, 
has  become  quite  generally  adopted  in  American  practice.  It  is,  of 
course,  unnecessary  to  enter  into  a  consideration  of  the  significance  of 
the  numbers  of  bacteria  determined  by  this  procedure  at  this  time. 

The  value  of  a  determination  of  the  numbers  of  bacteria  which 
are  able  to  develop  at  body  temperature  was  first  advanced  in  1892 
by  Wurtz.'^  In  1893  Matthews^  quite  clearly  demonstrated  the  dis- 
tinction in  the  bacterial  content  of  different  classes  of  polluted  and 
non-polluted  waters  by  the  use  of  lactose  agar  plates  incubated  at 
body  temperature,  thus  confirming  the  deductions  of  Wurtz.  Routine 
determinations  of  the  number  of  bacteria  producing  acid  fermenta- 
tion of  lactose  (colon  type)  on  plates  incubated  at  38°  to  40°  C.  have 
been  made  on  certain  classes  of  waters  since  1896  at  Lawrence,  and 
a  similar  procedure  has  been  adopted  by  a  large  number  of  water  bac- 
teriologists. The  value  of  counts  of  the  total  number  of  bacteria 
developing  at  38°  to  40°  C,  however,  appears  to  have  been  lost  sight 
of  in  the  rush  of  bacteriologists  to  study  the  acid-fermenting  types, 
until  Winslow  and  Niebecker^  in  1903,  after  extensive  investigations, 


Bacteria  Developing  at  Different  Temperatures     227 

arrived  at  exactly  the  same  conclusions  as  had  Matthews  ten  years 
earlier.  The  peculiar  advantage  which  such  determinations  have,  is 
that  results  may  be  obtained  in  18  to  24  hours,  or  one  to  three  days 
earlier  than  is  possible  with  plates  incubated  at  room  temperature. 

The  significance  of  the  numbers  of  bacteria  developing  on  plates 
incubated  at  temperatures  above  40°  C.  has  received  very  little 
study,  although  the  fact  that  such  bacteria  are  more  or  less  com- 
mon has  been  known  for  some  time.  In  1888  Gl()big'°  first  dem- 
onstrated that  certain  types  of  bacteria  capable  of  growing  at  tem- 
peratures of  50°  C.  or  higher  were  common  in  the  soil,  although 
Miquel  had  shown  the  existence  of  such  bacteria  some  five  years 
before.  In  1889-91  Miquel"  found  organisms  of  this  class,  which 
he  named  thermophylic  bacteria,  to  be  prevalent  in  polluted  waters, 
but  failed  to  detect  them  in  spring  waters.  Macfadyen  and  Blaxall'^ 
in  1894  demonstrated  the  presence  of  thermophylic  bacteria  in  hu- 
man feces,  and  Rabinowitschi'^  in  the  following  year  found  this 
type  of  bacteria  to  be  present  in  the  excrement  from  the  majority 
of  domestic  animals.  More  recently,  Houston, ''^  1898,  has  isolated 
similar  types  from  London  sewage  and  from  Thames  water,  and 
has  called  attention  to  their  significance  in  determining  the  extent 
of  pollution  of  a  water.  Tests  for  bacteria  of  this  type  included 
in  this  paper  were  made  on  plates  incubated  at  a  uniform  temper- 
ature of  50°  C. 

So  far  as  has  come  to  the  knowledge  of  the  writer,  no  comparative 
study  has  ever  been  made  of  the  numbers  of  bacteria  in  different 
classes  of  waters  which  will  develop  on  plates  incubated  at  30°  C, 
although  it  seemed  reasonable  to  believe  that  some  intermediate 
temperature  between  20°  and  40°  would  prove  favorable  for  the 
growth  of  a  large  number  of  bacteria  during  a  very  short  period 
of  incubation. 

Time  of  incubation. — The  time  allowed  to  elapse  betw'een  plating 
and  counting  varies  widely  in  different  laboratories,  depending  upon 
local  conditions  and  the  medium  employed.  Owing  to  the  slow 
development  of  bacteria  at  20°  C,  it  is  impossible  to  obtain  counts 
in  less  than  48  hours  on  plates  incubated  at  that  temperature. 
When  gelatin  is  employed,  it  is  usual  to  count  plates  on  the  second 
or  third  day,  the  value  of  the  determinations  being  obscured  by 


2  28  Stephen  DeM.  Gage 

liquefaction  when  longer  periods  of  incubation  are  followed.  It 
is  customary  to  incubate  agar  plates  three  to  four  days,  although 
in  some  laboratories  even  longer  periods  are  allowed  to  elapse  before 
the  plates  are  counted.  The  recommendation  of  the  Committee 
on  Standard  Methods,  that  a  uniform  period  of  incubation  of  48 
hours  be  employed,  has  been  quite  generally  adopted  by  users  of 
gelatin,  but  has  found  less  favor  among  users  of  agar.  At  Lawrence 
it  has  always  been  the  custom  to  allow  four  days  to  elapse  between 
planting  and  counting. 

With  the  use  of  higher  temperatures,  and  the  more  rapid  growth 
of  bacteria  at  such  temperatures,  it  becomes  possible  to  adopt  a 
shorter  period  of  incubation.  Since  it  is  extremely  desirable  that 
the  results  of  bacterial  determinations  be  available  at  the  earliest 
possible  moment,  the  writer  has  adopted  a  uniform  time  of  incu- 
bation of  24  hours  on  all  plates  grown  at  temperatures  higher  than 
20°  C. ;  consequently  the  numbers  of  bacteria  included  in  the  dis- 
cussion and  tables  beyond,  unless  otherwise  stated,  were  obtained 
on  plates  incubated  four  days  at  20°  C,  and  24  hours  at  30°,  40°, 
and  50°  C,  respectively. 

The  use  of  ratios  between  different  bacterial  counts. — In  comparing 
the  analytical  results  obtained  from  dififerent  waters,  or  from  different 
samples  of  the  same  water,  it  is  quite  usual  to  express  the  results 
of  such  comparison  as  the  ratio,  or  per  cent,  which  one  is  of  another. 
The  mathematical  expression  of  the  ratios  between  the  results  of 
different  determinations  is  less  common,  and  has  hitherto  been 
confined  to  the  chemical  side  of  the  analysis,  so  far  as  the  writer 
knows,  no  use  ever  having  been  made  of  the  ratios  between  the 
results  of  different  bacteriological  determinations  on  the  same  sam- 
ples. 

In  the  present  study  we  have  the  results  of  bacterial  counts  on 
agar  and  on  litmus-lactose  agar  plates  which  have  been  grown  at 
four  different  temperatures,  and  upon  which  the  number  of  bacterial 
colonies  have  been  counted  daily  until  the  maximum  number  of 
colonies  has  developed.  In  addition  to  the  determined  numbers  of 
bacteria  on  each  of  the  plates,  and  the  numbers  of  bacteria  which 
are  able  to  cause  acid  fermentation  of  lactose,  both  of  which  determi- 
nations may  have  a  place  in  the  interpretation  of  the  character  of  a 


Bacteria  Developing  at  Different  Temperatures     229 

water,  we  may  compute  the  ratios  between  each  pair  of  resuUs  on  the 
different  samples,  and  by  averaging  the  ratios  so  obtained  for  different 
classes  of  samples  we  may  ascertain  what  value  each  of  the  different 
ratios  may  have  in  indicating  the  quality  of  a  water.  As  a  con- 
sideration of  all  of  the  experimental  data,  and  of  all  the  possible 
ratios,  would  occupy  more  space  than  is  available,  many  of  the  data 
have  been  excluded,  and,  in  addition  to  a  consideration  of  the  num- 
bers of  bacteria  determined  on  agar  after  four  days'  incubation 
at  20°  C,  and  on  lactose  agar  after  24  hours'  incubation  at  30°,  40°, 
and  50°  C.  respectively,  and  the  numbers  of  bacteria  producing  red 
colonies  on  lactose  agar  at  the  same  temperatures,  only  the  follow- 
ing ratios  will  be  presented,  these  ratios  being  expressed  in  every 
case  as  the  per  cent  which  the  smaller  value  is  of  the  greater. 

1.  The  ratios  between  the  total  number  of  bacteria  determined 
on  agar  after  four  days'  incubation  at  20°  C,  and  the  numbers  of 
bacteria  determined  on  lactose  agar  after  24  hours'  incubation  at 
30°,  40°,  and  50°  C.  respectively. 

2.  The  ratios  between  the  total  number  of  bacteria  determined 
on  agar  after  four  days'  incubation  at  20°  C.  and  the  numbers  of 
bacteria  producing  acid  fermentation  on  lactose  agar  in  24  hours 
at  30,°  40,°  and  50°  C.  respectively. 

3.  The  ratios  between  the  numbers  of  bacteria  and  the  number 
of  acid-producing  bacteria  determined  at  each  temperature  as  above. 

Sources  oj  the  data  included. — The  data  used  in  the  discussion 
and  tables  have  been  obtained  in  part  by  recomputation  of  certain 
of  the  routine  results  obtained  during  the  past  eight  years  at  the 
Lawrence  Experiment  Station,  and  in  part  from  a  special  study 
of  the  relative  counts  of  bacteria  obtained  on  plates  incubated  at 
different  temperatures.  In  addition  to  the  incubators  regularly 
operated  at  20°  C.  and  40°  C,  we  have  one  spare  incubator,  which 
has  been  operated  part  of  the  time  at  30°  C.  and  part  of  the  time  at 
50°  C.  In  considering  the  significance  of  the  numbers  of  bacteria 
and  the  numbers  of  acid-producing  bacteria  determined  at  the 
different  temperatures,  it  is  therefore  necessary  to  divide  the  results 
into  two  series.  The  two  experiments  covered  much  the  same 
classes  of  waters,  and  had  determinations  of  bacteria  and  acid-pro- 
ducers at  20°  C.  and  at  40°  C.  common  to  both  experiments.     No 


230  Stephen  DeM.  Gage 

direct  comparison,  however,  was  possible  between  the  results  obtained 
at  30°  C.  and  50°  C,  and  we  are  forced  to  make  such  a  comparison 
indirectly  through  the  counts  at  20°  C.  and  40°  C.  It  should  be 
further  borne  in  mind  that,  while  the  series  containing  the  50°  C. 
counts  covered  a  wide  range  of  samples,  only  five  samples  were 
used  from  each  of  the  several  sources,  and  these  samples  were  all 
collected  within  a  limited  period  of  time.  For  this  reason  the  results 
of  this  series,  while  indicative  of  the  results  which  might  be  obtained 
in  practice,  should  not  be  taken  as  conclusive  evidence  of  the  value 
of  the  50°  counts.  On  the  other  hand,  the  results  obtained  in  the 
30°  series,  while  covering  a  smaller  range  of  waters,  were  obtained 
from  the  examination  of  over  400  samples  collected  over  a  period 
of  many  months. 

Information  bearing  on  the  subject  at  hand  has  also  been  drawn 
from  the  routine  analyses,  as  follows:  About  80  samples  from  15 
different  wells,  over  200  samples  of  sea  water  from  different  loca- 
tions in  Boston  Harbor,  and  nearly  1,000  samples  of  polluted  river 
water  upon  which  bacterial  counts  at  temperatures  of  20°  and  40° 
have  been  made,  the  counts  at  40°  being  on  litmus-lactose  agar,  and 
including  both  total  colonies  and  red  colonies,  determinations  of 
numbers  of  bacteria  and  B.  coli  in  over  300  samples  of  sewage  and 
effluents  from  sewage  filters,  and  in  about  3,700  samples  of  Merri- 
mack River  water  from  two  sources;  from  which  data  we  have 
been  able  to  determine  very  accurately  the  average  ratio  of  bacteria 
to  B.  coli  for  these  classes  of  samples,  and  in  the  river  water  results 
to  study  the  various  factors  which  have  caused  changes  in  the  num- 
bers of  bacteria  and  of  B.  coli,  and  the  ratios  between  the  two. 

Classification  of  waters  included  in  the  investigation. —  During 
the  various  investigations  17  different  classes  of  samples  have 
been  examined  at  one  time  or  another,  and  it  is  necessary,  for 
a  clear  understanding  of  the  tables,  that  a  brief  description  of  these 
classes  of  samples  should  be  inserted  at  this  point. 

Lawrence  Street  sewage  is  a  strong  domestic  sewage  collected  directly  from  one 
of  the  large  sewers  in  the  city  of  Lawrence. — Regular  sewage  is  sewage  from  the  same 
source  as  the  foregoing,  conveyed  to  the  Experiment  Station  through  about  a  mile 
of  2 . 5  inch  pipe,  and  differs  from  the  Lawrence  Street  sewage  mainly  in  the  fact  that 
the  grosser  particles  are  very  largely  broken  up  by  abrasion  and  by  passing  through 
the  pump. — Station  sewage  is  regular  sewage  diluted  with  a  small  proportion  of  Mer- 


Bacteria  Developing  at  Different  Temperatures     231 

rimack  River  water. — Septic  sewage:  Under  "septic  sewage"  has  been  included 
effluents  from  three  septic  tanks,  all  of  which  treat  regular  sewage. — Strained  sewage 
is  regular  sewage  from  which  a  portion  of  the  suspended  matter  has  been  removed 
by  straining  through  coal. — Intermittent  sewage  filters:  Samples  from  a  number  of 
intermittent  sand  filters  operating  at  low  rates  with  regular  sewage  or  with  station 
sewage  are  included  in  the  various  tables. — Trickling  filters:  Samples  from  two  differ- 
ent trickling  filters  operating  at  high  rates  vdth  regular  sewage  are  included. — Con- 
tact  filters:  Samples  from  three  different  contact  filters  have  been  used,  No.  175  oper- 
ating with  regular  sewage,  No.  176  with  strained  sewage,  and  No.  251  with  septic 
sewage. — Merrimack  River  water:  Samples  from  two  sources  have  been  used,  the 
source  labeled  "Intake"  including  samples  of  the  water  as  it  flows  upon  the  Law- 
rence city  filter.  Samples  labeled  "Canal"  are  the  river  water  as  received  at  the 
Experiment  Station  after  passing  through  the  North  Canal.  The  distance  from 
the  Experiment  Station  to  the  head  of  the  canal  is  about  one  mile,  and  that  from  the 
head  of  the  canal  to  the  Intake  of  the  City  Filter  about  one  mile,  no  sewage  entering 
the  river  or  canal  between  these  points. — Applied  216:  This  is  canal  water  which 
has  been  treated  with  alum  and  settled  before  being  applied  to  Filter  No.  216 — 
Water  filters:  "Filter  No.  216"  is  a  mechanical  filter  operating  at  a  high  rate  with 
the  above.  "Filters  Nos.  8,  220,"  and  the  "City  Filter"  are  slow  sand  filters  oper- 
ating at  low  rates,  the  city  filter  receiving  Merrimack  River  water  designated  "Intake," 
and  the  others  canal  water.  "Filters  Nos.  24J  and  244"  are  secondary  filters  oper- 
ating with  water  which  has  already  been  filtered. — Tap  water:  This  is  filtered  water 
from  the  Lawrence  city  filter,  after  being  stored  in  the  distribution  reservoir  and 
passing  through  the  city  service  mains. — Ponds:  Many  samples  from  two  large  ponds, 
both  used  for  water  supply,  have  been  examined.  The  watersheds  of  both  of  these 
ponds  are  under  sanitary  control,  but  both  are  used  more  or  less  for  pleasure  pur- 
poses in  the  summer,  and  hence  are  liable  to  occasional  contamination. — Driven 
wells:  The  samples  were  from  two  series  of  driven  wells  less  than  50  feet  deep  used 
as  water  supply. — Shallow  wells:  Samples  from  15  different  wells,  the  results  from 
both  good  and  polluted  wells  being  averaged  together.  In  tables  where  results  from 
two  different  wells  are  given,  "No.  i"  is  of  excellent  quality,  while  "No.  2"  is  badly 
polluted. — Springs:  Samples  from  two  different  springs,  both  of  good  quality,  are 
included. — Sea  waters  include  samples  collected  from  a  large  number  of  stations 
during  a  sanitary  survey  of  Boston  Harbor.  Most  of  these  samples  were  more  or 
less  polluted  with  sewage. 

Selective  action  of  different  temperatures. — In  considering  the 
value  of  counts  of  total  bacteria  and  of  acid-producing  bacteria, 
in  addition  to  the  relative  numbers  obtained  by  such  counts,  we 
should  know  the  selective  action  of  the  different  temperatures  upon 
the  bacterial  content  of  the  various  classes  of  waters.  We  have 
learned  by  these  experiments  that  nearly  all  of  the  bacteria 
capable  of  forming  colonics  at  20°  C.  will  manifest  themselves  in 
four  days,  this  being  also  true  of  the  acid-producing  bacteria.  At 
40°  C.  and  50°  C.  nearly  all  the  bacteria  and  acid-producers  capable 
of  developing  at  these  temperatures  are  shown  by  a  count  made  after 


232 


Stephen  DeM.  Gage 


24  hours.  At  30°  C,  however,  our  24-hour  counts  show  us  only 
30  to  50  per  cent  of  the  bacteria  which  would  be  able  to  produce 
colonies  if  the  period  of  incubation  were  increased  to  two  or  three 
days.  In  Table  i  is  shown  the  per  cent  of  samples  in  which  bac- 
teria and  acid-producing  types  occurred  in  waters  from  different 
sources  when  examined  by  the  various  methods.  The  results  of  deter- 
minations of  acid-producing  bacteria  at  20°  divide  themselves  nat- 
urally into  groups.  All  of  the  samples  of  sewage  and  effluents 
from  sewage  filters,  and  over  80  per  cent  of  the.  polluted  waters 
and  effluents  from  sewage  filters,  contained  bacteria  of  this  type 
the  percentage  for  shallow  wells  dropping  to  70,  for  pond  waters 
to  60,  and  for  deep  wells  to  40.  The  springs  differed  from  the  other 
relatively  pure  waters  in  that  all  samples  contained  acid-producing 
bacteria.  At  30°  C.  the  percentage  of  samples  showing  growth 
and  acid-producing  bacteria  is  generally  indicative  of  the  quality 
of  the  water,  becoming  less  as  the  quality  of  the  water  improves. 
The   same   rule   holds  for  the  determinations  made  at  40°  C,  the 

TABLE  I. 

Relative  Occurrence  of  Bacteria  and  of  Acid-Producing  Types  on  Plates  Incubated  ai  Dif- 
ferent Temperatures  with  Various  Classes  of  Waters. 


Per  Cent  of  Samples  Showing 

Growth  at 

Red  Colonies  at 

30°  c. 

40°  C. 

so°c. 

20°  C. 

30°  c. 

40°  c. 

50°  c. 

Sewage 

94 

100 

77 

61 

23 

100 

100 

100 

9S 

100 

100 

90 

83 

48 

80 

0 

30 

93 
100 
80 
60 
90 
100 
60 

55 

10 

20 

0 

0 

100 

100 

100 

100 

91 

94 

90 

83 
60 
70 
40 
100 

91 
96 

77 
36 
10 

lOO 

100 
100 
75 
100 
97 
83 
57 
20 

5° 

0 

30 

60 

Sewage  filters— trickling 

— contact. 

40 
40 
17 
70 
80 

"     — sand 

Merrimack  River 

Applied  216 

Filter  No.  216 

60 

Other  water  filters 

50 

Ponds: 

Shallow  wells 

Driven  wells 

Springs 

distinction  between  samples  known  to  have  been  polluted  recently, 
even  although  they  have  been  subjected  to  purification,  and  samples 
from  sources  whose  chance  of  pollution  is  more  remote,  being  espe- 
cially well  marked.  The  driven-well  waters  showed  entire  absence 
of  bacteria  growing  at  this  temperature.  The  distinction  between 
sewage  and  polluted  water  before  filtration,  and  the  effluents  from 


Bacteria  Developing  at  Different  Temperatures     233 

sewage  and  water  fillers,  is  shown  by  llie  smaller  per  eenl  of  samples 
from  the  filtered  sources  which  contain  bacteria  and  acid-producing 
organisms  capable  of  developing  at  50°  C.  Bacteria  of  these  types 
occurred  only  in  a  small  percentage  of  the  samples  from  ponds 
and  shallow  wells,  and  were  entirely  absent  from  samples  from  springs 
and  driven  wells. 

Numbers  0}  bacteria,  jo°  series. — In  Table  2  are  shown  the  aver- 
age numbers  of  bacteria  and  of  acid-producers  obtained  w  ilh  different 
waters  on  plates  incubated  at  20°  C,  30°  C,  and  40°  C.  From  the 
figures  in  this  table  it  is  seen  that  the  numbers  obtained  at  the  dif- 
ferent temperatures  agree  in  general  with  the  character  of  the  water 
under  examination.  The  counts  obtained  at  30°  were  larger  than 
those  at  40°  in  every  instance  when  dealing  with  polluted  water, 
but  there  was  little  difference  in  these  counts  when  dealing  with 
water  of  good  quality.  This  fact,  however,  would  be  an  advantage 
rather  than  otherwise,  since  the  use  of  30°  counts  would  give  us  a 
much  sharper  distinction  between  good  and  bad  waters  than  would 
counts  at  40°,  and  would  allow  this  distinction  to  be  made  in  a  min- 
imum time,  24  hours,  as  compared  with  two  to  four  days  required 
to  obtain  the  20°  counts.  A  rapid  method  of  determining  a  portion 
of  the  bacterial  content  of  waters  is  especially  desirable  when  deal- 
ing with  water  filters,  such  filters  being  controlled  largely  on  the 
basis  of  the  per  cent  of  the  bacteria  in  the  raw  water  which  they 
remove  and  the  number  of  bacteria  in  the  filtered  water.  From  the 
figures  in  the  table  we  find  the  percentage  removal  of  bacteria  by 
the  Lawrence  City  Filter  to  be  99.5,  99 -7,  and  96.7,  as  determined 
by  the  counts  at  20°,  30°,  and  40°  respectively,  and  the  removal 
of  acid-forming  organisms  to  be  99.1,  99 -4,  and  95.2  respectively. 
The  percentage  efficiency  of  Filter  No.  220  was  98.1,  96.0,  and 
93.8  respectively,  computed  from  the  total  bacteria  developing 
at  20°,  30°,  and  40°  C,  and  98.8,  98.6,  and  97.7  computed  from 
the  acid-forming  bacteria  developing  at  these  temperatures.  The 
removal  of  bacteria  by  Filter  No.  216  was  91.0,  96.1,  and  82.9 
respectively,  and  the  removal  of  acid-forming  bacteria  was  95.0, 
94.6,  and  82.2  respectively,  as  shown  by  the  figures  at  20°,  30°, 
and  40°  C.  In  other  words  the  percentage  removal  of  bacteria  and 
of  acid-formers,  as  determined  by  counts  at  20°  C.  after  four  days' 


234 


Stephen  DeM.  Gage 


incubation  and  at  30°  C.  after  24  hours'  incubation,  was  very  similar, 
and  although  the  numbers  of  bacteria  determined  at  30°  C.  were 
smaller  than  at  20°  C,  they  were  fully  as  significant.  The  substi- 
tution of  the  24-hour  count  at  30°  for  the  usual  20°  count  in  labora- 
tories connected  with  water  filtration  plants  would  result,  therefore, 
in  a  reduction  of  the  period  required  to  complete  a  bacterial  anal- 
ysis, without  causing  any  material  change  in  the  theories  upon  which 
the  interpretation  of  the  results  are  based.  The  value  of  counts 
at  40°  C.  made  at  the  same  time  as  counts  at  20°  or  30°  should  not 
be  underestimated,  since  the  group  of  bacteria  determined  at  this 
temperature  undoubtedly  represent  more  closely  the  pathogenic 
bacterial  content  of  the  water  than  is  the  case  with  counts  at  lower 
temperatures.  It  would  be  inadvisable  however,  to  employ  40° 
counts  exclusively  in  water  work  at  the  present  time,  until  we  have 
a  broader  understanding  of  their  complete  significance,  although 
such  counts  are  used  exclusively  in  some  laboratories  in  the  control 
of  milk  supplies.  The  average  results  of  counts  at  the  various  tem- 
peratures with  eight  different  waters  are  shown  in  the  following 
table : 

TABLE  2. 

Average  Number  or   Bacteria  and   Acid-Producers   Developing   at   20",  30",  and  40°  C.  with 

Different  Classes  of  Water. 


Canal 

Intake  .  .  .  . 
Applied  216 
Filter  216  .  . 
Filter  220  . . 
City  filter  . . 
Pond  No.  I . 
Pond  No.  2 . 


Bacteria  per  c.  c. 


o°C. 
4D. 


4,100 

5,000 

2,000 

180 

80 

23 
12 

17 


30"  C. 
24  Hr. 


450 

619 

203 

8 

18 

2 

o 

I 


40 


24  Hr. 


81 
122 
46 
7 
5 
4 
I 
I 


Acid-Producing  Bacteria 


20°  C. 
4D. 


940 

660 

537 

27 

II 

6 

2 

I 


30°  C. 
24  Hr. 


142 

173 

55 

3 

2 
I 

O 

o 


40°  c. 

24  Hr. 


44 
42 
28 

S 
I 
2 
o 
I 


Bacterial  ratios,  jo°  series. — In  Table  3  are  shown  the  various 
bacterial  ratios  for  the  determinations  at  20°,  30°,  and  40°  C.  Cer- 
tain distinctions  between  the  different  waters  are  brought  out  by 
these  ratios  which  do  not  appear  in  the  numbers  of  bacteria.  In 
the  first  two  columns  are  shown  the  per  cent  which  the  numbers 
of  bacteria  determined  at  30°  and  40°  after  24  hours'  incubation 
are  of  the  total  bacteria  determined  at  20°  after  four  days'  incuba- 


Bacteria  Developing  at  Different  Temperatures     235 

tion.  From  these  figures  we  see  that,  with  one  exception,  a  much 
greater  percentage  of  the  total  bacteria  are  determined  at  30°  for 
polluted  waters  than  for  the  pure  waters;  that  is  to  say,  the  dis- 
tinction between  pure  and  polluted  waters  is  emphasizx-d  by  the 
30°  counts.  On  the  other  hand,  the  counts  at  40°  materially  decrease 
such  a  distinction,  as  is  shown  by  the  fact  that  the  ratios  arc  greater 
for  the  good  waters  than  for  the  polluted  waters.  If  we  assume, 
however,  that  the  presence  of  bacteria  capable  of  rapid  growth  at 
40°  is  an  indication  that  the  water  contains  disease-producing  bac- 
teria, the  fact  that  the  proportion  of  such  bacteria  is  greater  in  the 
purer  waters  than  in  the  waters  known  to  be  seriously  polluted 
would  signify  that  our  filtered  waters  were  not  of  such  excellent  qual- 
ity as  their  low  bacterial  content  would  indicate,  which  supposition 
is  well  worth  further  study. 

No  such  lesson  is  apparent  in  the  ratios  between  the  total  bacteria 
at  20°  and  the  acid-producing  bacteria  at  30°  and  40°,  the  values 
for  the  raw  waters  and  filtered  waters  being  much  the  same.  A  sharp 
distinction  is  noted  between  the  ponds  and  the  other  samples  in  the 
30°  values,  although  this  distinction  does  not  appear  in  the  40° 
values.  The  ratios  between  the  bacteria  growing  at  each  tempera- 
ture and  the  number  of  acid-formers  at  that  temperature  appear 
to  be  of  little  value  in  distinguishing  between  the  different  waters. 
As  will  be  shown  later,  the  chief  use  of  these  ratios  seems  to  be  in 
locating  errors  and  abnormal  values  in  the  other  ratios  and  in  the 
counts  from  which  they  are  computed. 

TABLE  3. 
Bacterial  Ratios  for  Different  Classes  of  Waters,  20°,  30°,  akd  40'  C.  Series. 


Canal 

Intake 

Applied  2 16 
Filter  216  .  . 
Filter  220  .  . 
City  filter  . . 
Pond  Xo.  I. 
Pond  No.  2. 


Ratio  between 

Total  Bacteria 
a  t  20°  and 
Bacteria  De- 
veloping at 


30"  C. 


12.20 
12.38 
10.  IS 

4  45 
22.50 

8.70 
0.00 

5  90 


40°  c. 


97 
44 

so 
80 

2S 


17    40 
8.33 

s  90 


Ratio  between 
Total  Bacteria 
at  20°  and 
Number  of 
A  c  i  d-Produc- 
ing  Bacteria  at 


30- 


3.46 

3  46 
2  75 
1.67 
2.50 

4  35 
0.00 
0.00 


40~ 


c. 


.07 

84 

40 

78 

is 

70 

.00 

00 


Ratio  l-)etween  Number  of 
Bacteria  at  Each  Tem- 
perature and  Acid-Pro- 
ducinK  Bacteria  at  That 
Temperature 


C. 


23 
13 

27 

IS 

14 

26 

16 

6 


30-C. 


32 
28 

27 
38 
ti 

50 
o 

o 


40- 


54 
34 
6i 
7« 
20 
SO 
o 
100 


236  Stephen  DeM.  Gage 

Numbers  0}  bacteria,  50°  series. — The  average  numbers  of  bac- 
teria and  the  numbers  of  acid-producers  determined  at  20°,  40°, 
and  50°  on  five  samples  each  from  26  different  sources  are  shown 
in  Table  4.  In  general  the  numbers  of  bacteria  at  20°  and  40° 
and  the  numbers  of  acid-producers  determined  at  those  tempera- 
tures, are  large  or  small  as  the  water  is  polluted  or  non-polluted, 
confirming  the  findings  previously  discussed  under  the  30°  series. 
The  most  significant  results  are  those  obtained  for  the  two  shallow 
wells.  The  number  of  bacteria  determined  at  20°  is  higher  in  Well 
No.  I  than  in  Well  No.  2.  Well  No.  2  is  in  a  thickly  settled  communi- 
ty with  vaults  and  cesspools  in  close  proximity,  while  Well  No.  i,  sit- 
uated in  an  open  field  upon  the  top  of  a  hill,  is  removed  from  any 
chance  of  pollution.  Chemical  analyses  extending  over  a  period 
of  some  years  indicate  that  Well  No.  i  is  free  from  pollution,  and 
that  Well  No.  2  is  seriously  polluted.  The  sanitary  survey  and  chemi- 
cal analyses  are  confirmed  by  the  bacterial  count  at  40°  and  by 
the  numbers  of  acid-producers  developing  at  20°  and  40°,  showing 
that  the  large  numbers  of  bacteria  determined  at  20°  in  Well  No.  i 
are  of  a  harmless  character. 

The  numbers  of  bacteria  and  the  acid-formers  determined  at 
50°  C.  confirm  the  results  of  determinations  at  20°  and  40°,  but 
the  distinction  between  different  classes  of  waters  is  more  marked 
than  by  determinations  at  the  lower  temperatures.  It  is  noticeable 
that  the  50°  bacteria  in  the  effluents  from  the  Contact  Filters  Nos. 
175  and  176  were  higher  than  in  the  regular  sewage  which  was  ap- 
plied to  those  filters,  and  the  numbers  in  the  effluent  of  Contact 
Filter  No.  251  were  larger  than  in  the  septic  sewage  with  which  it 
was  operated.  On  the  other  hand,  the  numbers  in  the  effluents 
from  the  trickling  filters  were  small,  and  this  type  of  bacteria  were 
either  entirely  lacking  or  present  in  insignificant  numbers  in  the 
effluents  from  the  sand  filters.  The  distinction  between  the  river 
water  and  the  filtered  waters  is  not  very  well  marked,  but  a  class  dis- 
tinction between  the  river  water  and  the  filtered  river  waters,  and  the 
ponds,  wells,  and  springs,  is  indicated  by  the  entire  absence  of  this 
type  of  organisms  in  the  latter  class  of  waters.  The  occurrence 
of  50°  acid-producing  bacteria  is  also  significant,  this  type  of  organ- 
isms being  absent  from  the  effluents  from  three  out  of  four  of  the 


Bacteria  Developing  at  Different  Temperatures      237 

intermittent  sewage  filters,  and  practically  absent  from  the  fourth, 
while  they  were  present  in  greater  or  less  numbers  in  the  sewages 
and  in  the  effluents  from  the  contact  and  trickling  filters  through 
which  the  sewage  passed  more  rapidly.  The  presence  of  such  small 
numbers  of  this  type  of  bacteria  in  the  polluted  river  water,  and 
of  similar  numbers  in  the  effluents  from  the  primary  water  filters, 
cannot  be  accounted  for  at  the  present  time. 


table  No.  4. 

Average  Number  of  Bacteria  and  Acid -Producers  Developing  at  20°,  40°,  and  50°  C.  with 

Different  Classes  of  Waters. 


Regular  sewage 

Station  .sewage 

Septic  sewage   

Sand  Filter  No.  i 

"2 

4 

"9 

Trickling  Filter  No.  13s 

"        "      136 

Contact  Filter  No.  175. 

"     176. 

"    251. 

Canal 

Intake  

Applied  216 

Filter  No.  8 

Filter  No.  216 

Filter  No.  243 

City  filter 

C'ity  water 

Pond  No.  I   

Pond  No.  2  

Driven  wells 

Shallow  Well  No.  i 

Shallow  Well  No.  2 

Spring  No.  i 

Spring  No.  2 


Bacteria  per  c.c. 


20°  C. 
4D. 


,900,000 

,676,000 

485,000 

1.640 

35 

1.300 

670 

15.500 

23,300 

146.600 

380,000 

306,000 

16,400 

16,900 

2,8oo 

32 
7IS 

62 
150 

64 

27 
71 
41 

1,000 

S07 

49 
80 


40"  C. 
24  Hr. 


So°C. 
24  Hr. 


557. 500 

7.700 

360,000 

29.500 

126.500 

410 

1.375 

2 

4 

0 

130 

I 

170 

2 

1.730 

154 

2,030 

54 

26, 100 

8,300 

59.300 

8,000 

89,600 

485 

112 

5 

207 

4 

212 

2 

3 

I 

170 

2 

I 

0 

22 

I 

5 

I 

I 

0 

8 

0 

0 

0 

2 

0 

72 

0 

0 

0 

2 

0 

Acid-Producing  Bacteria 


20"  C. 
4D. 


1,940.000 

1,032,000 

241,000 

2,360 

29 

345 

1,045 

15,200 

16,000 

112,400 

292,000 

193,000 

6,700 

2,500 

1,650 

6 

259 

16 

14 
II 

8 
30 

o 

3 

82 

6 

8 


40°  C. 
24  Hr. 


346,000 

283,000 

90,000 

1. 195 

2 

119 

154 

1.360 

1,180 

22,700 

45.000 

46,000 

87 

134 

66 

I 

101 

o 

17 

3 

I 

S 
o 
I 
55 
o 
2 


So'C. 
24  Hr. 


4.400 

24,900 

240 

I 

o 

o 

o 

100 

20 

8,000 

8.000 

200 

2 
2 
I 
I 
1 
O 
I 

o 
o 
o 
o 
o 
o 
o 


Bacterial  ratios,  jo°  series. — The  bacterial  ratios  for  the  ditTercnt 
waters  included  in  the  50°  series  are  shown  in  Table  5.  In  general 
the  20°-40°  bacteria  ratios  and  the  ratios  between  the  20°  bacteria 
and  the  40°  acid-producers  were  much  greater  for  the  sewage  and 
the  effluents  from  sewage  filters  than  for  the  other  waters,  although 
there  are  a  few  exceptions  to  this  rule.  The  20°-40°  ratios  for  the 
polluted  river  water  in  each  case  were  much  less  than  the  correspond- 
ing ratios  for  Applied  216  and  for  the  effluents  from  Water  niters 
No.  8,  2x6,  and  the  City  Filter,  indicating  that  the  removal  of  the 


238 


Stephen  DeM,  Gage 


ordinary  water  bacteria  by  coagulation  and  sedimentation,  and  by 
filtration,  is  greater  than  is  the  removal  of  bacteria  capable  of  devel- 
oping at  40°,  as  previously  noted  in  the  discussion  of  the  30°  series. 
The  peculiar  significance  in  the  ratios  between  the  bacteria  and  the 
acid-formers  at  20°  appears  to  be  in  the  much  larger  ratios  obtained 
for  sewages  and  the  effluents  from  sewage  filters  than  for  the  other 
waters  examined.  This  distinction  does  not  hold  true  for  the  40° 
and  50°  bacteria-acid-producing-organism  ratios,  the  high  and  low 
values  being  distributed  among  all  classes  of  waters. 

TABLE  5. 

Bacterial  Ratios  for  Different  Classes  of  Waters,  20°,  40°,  and  50°  C.  Series. 


Regular  sewage 

Station  sewage 

Septic  sewage 

Sand  Filter  No  i 

"         "        "2 

4 

'9 

Trickling  Filter  No.  135, 

'     136. 

Contact  Filter  No.  1 75 . . . 
176.., 

251... 

Canal 

Intake 

Applied  216 

Filter  No.  8 

"        "     216 

"    243 

City  filter 

City  water 

Pond  No.  I 

"        "    2 

Driven  wells 

Shallow  Well  No.  i 

"  "      "2 

Spring  No.  i 

"  "    2 


Ratio  between 
Total  Bacteria 
at  20°  and 
Bacteria  De- 
veloping at 


40 


19.00 
21.50 
26.00 
S3. 80 
II  .40 
10.00 

25.30 

II .  20 

8.70 

17.70 

15.20 

29.30 

0.68 

I.  22 

7.56 

9.40 

23.80 

1. 61 


70 
80 
70 

25 
00 
20 


14.20 
0.00 
2.50 


So°C. 


o 

I 

o 

I 

o 

o. 

o 

o 

o. 

5- 
2 . 
o. 
o. 
o. 
o. 

3 
o. 
o. 
o. 

I  56 

0.00 
00 
00 
00 
00 
00 
00 


23 

80 
08 

22 

00 
08 
30 

95 
23 
70 
10 
IS 
03 
02 

07 
12 
28 
00 
67 


Ratio  between 
Total  Bacteria 
at  20°  and 
Number  of 
Acid  -  Produc- 
ing Bacteria  at 


40°  C. 


II  .60 

16.90 

18.50 

72,80 

5 -70 

9.  20 

23.00 

8.80 

510 

15   50 

II  .50 

IS  00 

0-53 

0.79 

2.36 

312 

14.  10 

0.00 

"•35 

4.70 

3    70 

7.40 

0.00 

o.  10 

10.80 

0.00 

2.50 


50 


015 

1.49 

COS 

0.61 

0,00 

0.00 

0.00 

0.65 

0.09 

so 

10 

07 

01 

01 

04 

12 

14 

00 

67 


1.56 
0.00 
00 
00 
00 
00 
00 
00 


Ratio  between  Number  01 
Bacteria  at  each  Tem- 
perature and  Acid-Pro- 
ducing Bacteria  at  That 
Temperature 


20°  C. 


48 
62 

so 

83 
27 

98 
69 
76 

75 
63 
41 
15 
59 
19 
36 
26 
9 
17 
30 
42 
00 
00 
16 

12 
10 


40 


62 

79 
71 
87 
SO 
92 
91 
78 
S8 
87 
76 
51 
78 
65 
31 
33 
59 
00 
77 
60 
100 

63 
00 
50 
76 
00 
100 


SO 


57 
84 
59 
50 
00 
00 
00 
65 
37 
96 

100 
41 
40 
SO 
50 

100 
SO 
00 

100 

100 
00 
00 
00 
00 
00 
00 
00 


Bacterial  determinations  at  20°  and  40°  on  polluted  waters. — In 
addition  to  the  results  obtained  in  the  30°  and  50°  series  previously 
discussed,  we  have  somewhat  more  extended  information  regarding 
the  relation  between  the  numbers  of  bacteria  developing  at  20°  and 
at  40°  C.  Throughout  1905  both  total  colonies  and  red  colonies 
were  counted  on  all  litmus-lactose  agar  plates.  Comparative  counts 
are  thus  available  on  some  200  samples  of  sea  waters,  and  on  samples 


Bacteria  Developing  at  Different  Temperatures     239 

collected  at  least  three  times  a  week  throughout  the  year  from  four 
different  polluted  sources;  these  being  Merrimack  River  at  the  Intake 
of  the  City  Filter,  and,  from  the  North  Canal  at  the  Experiment 
Station,  river  water  which  has  been  treated  by  coagulation  and 
sedimentation,  and  river  water  in  which  the  pollution  has  been  in- 
creased by  the  addition  of  more  sewage.  The  samples  from  the 
Intake  and  Canal  showed  very  similar  results,  taken  month  by 
month,  as  is  shown  in  the  foregoing  tables,  and  for  this  reason  the 
Intake  samples  have  been  omitted.  The  samples  of  sea  water  exhibit 
certain  peculiarities,  and  the  samples  from  the  three  other  sources, 
while  similar  in  character,  represent  different  degrees  of  pollution 
and  through  them  we  may  gain  some  insight  into  the  relative  fluc- 
tuations in  the  bacteria  and  in  the  bacterial  ratios. 

As  the  sea  waters  included  samples  of  varying  degrees  of  pol- 
lution, some  division  of  the  samples  into  groups  becomes  advisable. 
A  number  of  methods  of  grouping  these  samples  have  been  tried, 
the  most  satisfactory  from  the  standpoint  of  the  subject-matter  of 
this  paper  being  to  place  all  samples  having  similar  numbers  of 
bacteria  in  one  group,  and  to  average  all  the  results  in  each  group. 

The  results  shown  in  Table  6  have  been  obtained  in  this  manner, 
from  which  it  is  seen  that  the  average  numbers  of  bacteria  at  20°, 
the  numbers  at  40°,  and  the  numbers  of  B.  coli — i.  e.,  acid-producers 
show  a  similar  increase  until  the  numbers  of  bacteria  reach  5,000 
per  c.c,  when  the  40°  bacteria  and  the  B.  coli  drop  to  XQvy  low  num- 
bers and  again  increase  gradually  with  increasing  numbers  of  bac- 
teria. The  ratios  between  the  20°  and  40°  bacteria  show  a  corre- 
sponding increase  until  the  numbers  of  bacteria  reach  1,000  per  c.c, 
a  decrease  occurring  when  the  numbers  of  bacteria  are  between 
1,000  and  5,000,  and  the  ratios  becoming  extremely  small  as  the 
numbers  of  bacteria  increase  above  5,000.  The  same  peculiarity 
is  noted  for  the  B.  coli  ratios,  with  the  exception  that  the  ratio  for 
an  average  bacterial  content  below  100  is  greater  than  the  ratio 
for  a  bacterial  content  between  100  and  500.  The  ratios  between 
the  40°  bacteria  and  the  B.  coli  are  fairly  uniform,  fluctuating 
between  57  and  83.  The  reasons  for  the  peculiarities  above  noted 
cannot  be  assigned  without  a  careful  study  of  the  sources  of  the 
various    samples    and   a   consideration   of   all    the    various   factors 


240 


Stephen  DeM.  Gage 


influencing  the  character  and  quantity  of  the  bacterial  content,  all  of 
which  will  be  reported  elsewhere  by  another  department  by  whom 
these  samples  were  collected. 

TABLE   6. 

Numbers  of  Bacteria  Determined  at  20°  and  40°  C,  and  the  Corresponding  Bacterial 

Ratios  on  Samples  of  Sea  Water. 


Bacteria  per  c.c. 


Less  than  100 

Between  100  and  500 

"        500  and  1,000 

"         1,000  and  5,000.  .  . 

"         5,000  and  10,000.  . 

10,000  and  50,000. 

Over  50,000 


20°  C. 


Bacteria 
per  c.c. 


78 

370 

700 

2,500 

5,800 

19,400 

700,000 


40°  C. 


Bacteria 
per  c.c. 


6 

30 

170 

223 

5 

30 

86 


Acid- 
Producers 


5 

17 

no 

15s 

4 

19 

60 


Ratio  of 


20°  Bac- 
teria to  40° 
Bacteria 


7.7 
I 


24 


20"  Bac- 
teria to  40° 

Acid- 
Producers 


6.4 
4.6 
iS-7 
6.2 
o.  I 
o.  I 


40"  Bac- 
teria to 
Acid- 
Producers 


83 
57 
6s 
69 
80 
63 
70 


The  monthly  averages  of  the  determinations  of  bacteria  at  20° 
and  40°  C,  and  the  corresponding  bacterial  ratios  on  all  samples 
of  the  canal  water,  Applied  216  and  Applied  219,  are  shown  in 
Tables  7,  8,  and  9.  Comparing  the  canal  results  with  those  from 
Applied  216,  the  yearly  averages  show  us  that  treatment  of  the  canal 
water  by  coagulation  and  sedimentation  removed  about  one-half 

TABLE  7. 

Canal:    Average  Monthly  Numbers  of  Bacteria  at  20°  C,  at   40°  C,  and  of  B.  coli,  and  the 

Bacterial  Ratios. 


1905 


January 

February. .  . . 

March 

April 

May 

June 

July 

August 

September  . . 
October  .... 
November. . . 
December . .  . 
Average 


40° 

c. 

20°  c. 

Bacteria 

20-40   C. 
Bacteria 

Bacteria 

2o°-B.coli 

per  c.c. 

Bacteria 
per  c.c. 

B.  coli 
per  c.c. 

Ratio 

Ratio 

6.600 

114 

80 

1-73 

1 .21 

9,800 

143 

98 

1 .46 

1. 00 

5,700 

107 

63 

1.87 

I.  II 

2,300 

46 

5i 

2  .00 

1-43 

2,600 

64 

40 

2.46 

I   54 

8,600 

139 

81 

1.61 

0.94 

3.800 

iSi 

86 

3  96 

2.26 

7,100 

355 

189 

5  .00 

2.66 

14.200 

221 

155 

I.S6 

1 .09 

25,600 

647 

160 

2. S3 

0.63 

7,900 

175 

115 

2.21 

1-45 

6,300 

132 

97 

2 .  10 

1-54 

8,400 

191 

100 

2.37 

1. 41 

Bacteria 

4o°-B.  coli 

Ratio 


70 
69 
59 
72 
63 
58 
57 
53 
70 

25 

66 
74 
61 


of  the  bacteria  contents  of  the  water.  On  the  other  hand,  the  ratios 
show  us  that  the  removal  of  bacteria  capable  of  growing  at  40°  and 
of  B.  coli  was  less  than  that  of  the  total  bacteria.     Furthermore, 


Bacteria  Developing  at  Different  Temperatures     241 

while  the  fluctuation  in  the  numbers  of  total  bacteria,  of  bacteria 
growing  at  40°,  and  of  B.  coli,  was  less  in  the  Applied  216  than  in 
the  canal,  the  fluctuation  in  the  ratios  between  these  numbers  was 
very  much  greater. 

Comparing  the  canal  with  Applied  219,  we  find  that  by  adding 
a  small  proportion  of  sewage  to  the  water  we  have  increased  our  bac- 
terial content,  as  shown  by  higher  values  on  all  three  determinations; 

table  8. 

Applied  216:     Average  Monthly  Numbers  of  Bacteria  at  20°  C,  at  40"  C,  and  of  B.  coli.  and 

THE  Bacterial  Ratios. 


1905 


January  .  .  .  . 
February.  .  . 

March 

April 

May 

June 

July 

August 

September  . . 
October.  .  . . 
November.  . 
December. . 
Average 


40° 

C. 

20°  c. 
Bacteria 

20''-40''C. 

Bacteria 

Bacteria 
20"- b.  coli 

per  c.c. 

Bacteria 
per  c.c. 

B.  coli 
per  c.c. 

Ratio 

Ratio 

S.ooo 

69 

39 

1.28 

0.78 

7.900 

76 

46 

0.96 

O.S9 

2,300 

44 

24 

1. 01 

1 .04 

1,500 

25 

18 

1.67 

1 .  20 

i,6oo 

39 

23 

2   43 

I   43 

5.100 

105 

66 

2.06 

1.29 

1,000 

S8 

27 

S.80 

2.  70 

1,400 

104 

63 

7-45 

4.50 

4.S00 

223 

162 

4-95 

3  60 

8,600 

149 

96 

1-74 

I .  It 

4,800 

107 

71 

2.20 

I   48 

3,800 

72 

43 

1.88 

I   "3 

4,000 

89 

57 

2.86 

I   74 

Bacteria 

4o<'-B.  coli 

Ratio 


47 
63 
SS 
72 
59 
S8 
47 
61 
73 
64 
66 
60 
60 


but  by  comparing  our  ratios  we  find  that  we  have  increased  the  class 
of  bacteria  developing  at  40°,  in  which  must  be  included  the  disease 
germs  in  a  much  larger  proportion  than  we  have  increased  the  total 
bacterial  content.  The  fluctuation  in  the  numbers  of  the  difi"erent 
classes  of  bacteria  and  the  fluctuation  in  the  ratios  between  these 

table  9. 

Applied  219:     Average  Monthly  Numbers  of  Bacteria  at  20°  C,  at  40°  C,  and  of  B.  coli.  and 

THE  Bacterial  Ratios. 


1905 


May 

June 

July 

August 

September. . 
October .... 
November.  . 
December. . 
Average 


4o» 

c. 

20°  c. 

Bacteria 

2o''-4o'' 
Bacteria 

Bacteria 
20°-iB.  coli 

per  c.c. 

Bacteria 
per  c.c. 

B.  coli 
per  c.c. 

Ratio 

Ratio 

20,200 

S07 

339 

2.47 

I  67 

12,800 

260 

xo8 

2.03 

•    S4 

17,800 

850 

828 

4.76 

4  65 

8,800 

537 

251 

6.  10 

2  8s 

44,700 

616 

357 

1.38 

0  80 

46,400 

1034 

595 

2.21 

1.2S 

28,200 

1746 

791 

6.20 

2   80 

26,000 

778 

531 

2.99 

a  04 

25.600 

791 

486 

3  52 

3  20 

Bacteria 

40° -fi.  cdi 

Ratio 


67 
76 
07 
48 
S« 
58 

<A 
6s 


242 


Stephen  DeM.  Gage 


numbers  correspond  fairly  well  with  the  probable  difference  in  the 
character  of  the  two  waters.  In  other  words,  our  ratios  indicate 
that  both  Applied  216  and  Applied  219  were  more  dangerous  from 
a  sanitary  standpoint  than  the  difference  between  their  total  bacterial 
counts  and  the  count  on  the  canal  water  would  indicate. 

Bacteria-B.  coli  ratios  on  Merrimack  River  water. — We  will  now 
consider  the  ratio  between  the  numbers  of  bacteria  and  the  numbers 
of  B.  coli,  this  being  the  one  of  the  new  factors  here  introduced  upon 
which  we  have  the  most  extensive  data.  Routine  determinations 
of  bacteria  and  B.  coli  have  been  made  on  samples  of  Merrimack 
River  water  from  the  Intake  during  a  period  of  seven  years,  and  on 
samples  from  the  canal  during  a  period  of  eight  years.  The  average 
monthly  numbers  of  bacteria  and  B.  coli  in  samples  from  these  two 
sources  have  been  published  annually  in  the  reports  of  the  Massa- 
chusetts State  Board  of  Health,  and  it  is  unnecessary  to  reproduce 
them  here.  The  ratios  between  the  bacteria  and  B.  coli  computed 
from  the  monthly  averages  for  these  two  sources  are  shown  in  Tables 
10  and  II.  The  use  of  the  monthly  averages  tends  to  eliminate 
abnormal  values  and  fluctuations,  and  to  give  us  results  which  are  very 

TABLE    10. 

Average  Monthly  Bacteria-B.  coli  Ratios  on  Samples  from  the  Merrimack  Ri  ver  at  the 
Intake  of  the  City  Filter,  1899-1005,  inclusive. 


January 

February  

March 

April 

May 

June 

July  

August 

September  .  .  .  . 

October 

November 

December  .  . .  . 
Average  .  . 
Maximum 
Minimum. 


1899 

1900 

1901 

1902 

1003 

1904 

1905 

Av 

0.57 

0.69 

1 .64 

0.  29 

c.  26 

0.61 

0.71 

0 

0 

.S.3 

0 

47 

.s 

28 

0 

61 

0 

33 

0 

71 

0 

84 

0 

0 

,SO 

6 

.SO 

0 

7« 

0 

44 

0 

98 

0 

57 

0 

95 

I . 

0 

41 

I 

.SO 

2 

4.S 

0 

.S8 

I 

09 

0 

44 

Ob 

I . 

I 

10 

I 

■ib 

I 

4.S 

0 

«,S 

I 

41 

I 

71 

37 

I. 

I 

S7 

I 

48 

0 

7.S 

2 

40 

0 

7« 

I 

25 

0 

01 

I . 

I 

07 

I 

■Ii 

0 

61 

0 

43 

I 

78 

I 

56 

46 

I . 

0 

16 

I 

.SI 

I 

06 

I 

O.S 

I 

65 

0 

97 

30 

1. 

0 

».^ 

0 

03 

0 

SO 

1 

07 

0 

37 

0 

66 

40 

0. 

0 

24 

0 

22 

0 

•S.S 

0 

53 

0 

44 

I 

66 

10 

0. 

0 

6i 

I 

86 

4 

07 

I 

02 

I 

32 

0 

70 

84 

I. 

I 

4,S 

I 

O.S 

0 

16 

I 

03 

0 

66 

0 

64 

06 

0. 

0 

81 

I 

6S 

I 

44 

0 

02 

0 

92 

0 

96 

17 

I . 

I 

07 

6 

.SO 

4 

07 

2 

40 

I 

7« 

I 

71 

84 

6. 

0 

16 

0 

22 

0 

16 

0 

20 

0 

26 

0 

44 

0 

71 

0. 

68 
97 
47 
01 
35 
30 
33 
08 
82 
67 
76 
99 
12 
30 
16 


nearly  normal,  and  the  fluctuations  occurring  from  month  to  month 
must  be  considered  normal  fluctuations.  These  fluctuations  from 
the  normal  and  the  effect  of  various  factors  in  producing  normal 
fluctuations  will  be  considered  later. 

The  numbers  of  bacteria  and  of  B.  coli  in  the  Intake  samples 
have  usually  been  somewhat  greater  than  those  in  the  canal  samples. 


Bacteria  Developing  at  Different  Temperatures     243 

A  study  of  the  bactcria-5.  coli  ratios  reveals  that  those  ratios  also 
have  been  constantly  higher  for  the  Intake  samples  than  for  the  canal 
samples,  and  that  there  has  been  a  greater  uniformity  in  the  bacte- 
rial content  of  the  Merrimack  River  water  after  it  has  passed  through 
the  canal  and  the  short  distribution  pipes  to  the  experimental  filters 
at  the  Experiment  Station,  taking  the  results  year  by  year,  and 
month  by  month,  than  was  the  case  with  the  same  water  as  it  was 
applied  to  the  Lawrence  City  Filter. 

The  average  ratio  on  all  samples  collected  from  the  Intake  during 
the  entire  seven  years  was  1.12,  the  lowest  yearly  average  being 
0.81,  in  1899,  and  the  highest  1.63,  in  1900.  The  lowest  monthly 
average  occurred  in  August,  1899,  ^"d  again  in  December,  1901, 
when  the  ratio  was  0.16,  and  the  highest  monthly  average,  6.30, 
occurred  in  March,  1900.  The  least  variation  in  the  monthly  ratios 
for  any  one  year  occurred  in  1904,  the  difference  between  the  high- 
est and  lowest  values  in  that  year  being  1.27.     The  greatest  varia- 

TABLE  n 
Average  Monthly   Bacteria — B.  coli    Ratios  on    Sauples  from   the  Merrimack  River    from 

THE  North  Canal,  i8q8-ioo.s  Inclusive. 


January   

February 

March 

April 

May   

June 

July 

August 

September  .  .  . 

October 

November.  .  .  . 

December 

Average.  . 
Maximum 
Minimum 


1898 


.80 
•59 
•  4.S 
.20 
70 
■56 

.  12 
.  12 

■75 
■55 
07 
65 
07 
.  12 
■45 


1899 


0.83 
0.45 
o.  20 
0.65 
0.82 
0.97 

2.  50 
0.78 
0.41 
O.  I  T 
0.62 
0.93 

O  .80 

2  .  50 
O.  II 


1900 


0.47 

O  .60 

0.61 

o.  70 

0.98 

1.96 
2-59 

1.08 
0.71 


46 

24 

53 
.08 
59 
46 


1901 


2.56 
2  .42 
1. 41 
1 .  10 
0.83 
0.44 


47 
16 

32 
08 

27 
31 
95 
56 
08 


1902 


0.52 
0.61 
o.  14 
1.86 
1-37 
0.60 
043 
I  94 
0.97 
0.91 
098 
0.66 
0.92 
1.94 
o.  14 


1903 


0.39 

0.44 

0.61 

06 

95 


65 
90 
87 
57 
70 
40 
49 
75 
40 
39 


1904 


95 
22 

37 
50 
81 

93 

il 

88 

1 .00 
0.86 
038 

1 .01 
232 
0.37 


lOOS 


21 

00 
II 

43 
54 
94 
26 
66 

OQ 

63 
45 
54 
41 
66 

63 


Average 


0,97 
0.92 
0.62 
1 .06 
I  01 
0.88 

1  57 
'43 
0.96 
o.  56 

O   90 

O    81 

o  98 

2  66 
0.08 


tion  in  the  monthly  ratios  in  any  year  occurred  in  1900,  when  the 
difference  between  the  highest  and  lowest  ratios  was  6.08. 

The  average  ratio  on  all  samples  from  the  canal  during  the  entire 
period  of  eight  years  was  0.98,  the  lowest  yearly  average  being 
0.80,  in  1899,  and  the  highest  1.41,  in  1905.  The  lowest  monthly 
ratio  occurred  in  October,  1901,  when  the  ratio  was  0.08,  and  the 
highest  occurred  in  August,  1905,  when  the  ratio  was  2.66.  The 
least  variation  in  the  monthly  ratios  during  any  one  year  occurred  in 


244  Stephen  DeM.  Gage 

1903,  the  difference  between  the  highest  and  lowest  values  being 
1. 01,  and  the  greatest  variation  in  the  monthly  ratios  occurred 
in  1 901,  the  difference  between  the  highest  and  lowest  values  being 
2.48. 

Normal  ratios. — That  the  labor  of  computing  the  ratios  between 
the  various  bacterial  constituents  is  repaid  by  the  additional  informa- 
tion acquired  as  to  the  character  of  the  water,  is  clearly  demonstrated 
in  the  preceding  chapters.  The  bacterial  ratios  may  also  be  made 
to  serve  as  a  check  upon  the  bacterial  determination,  and  by  point- 
ing out  fluctuations  in  the  bacterial  contents  of  different  samples 
of  water  from  the  same  sources,  which  would  otherwise  have  passed 
unnoticed,  indicate  the  direction  in  which  further  investigations 
must  be  made  to  convert  the  hitherto  unexplainable  discrepancies 
between  analytical  results  into  significant  facts.  In  order  that  we 
may  have  a  clear  understanding  of  the  significance  of  the  different 
ratios,  and  of  the  relative  numbers  from  which  they  are  computed, 
it  is  important  that  we  should  know  what  are  the  normal  ratios 
for  different  classes  of  waters.  The  determination  of  the  normal 
value  for  any  set  of  variable  characters  is  distinct  from  the  determina- 
tion of  the  average  of  those  characters,  and  consists  in  arranging 
the  individual  values  in  groups,  each  group  containing  all  values 
which  are  between  certain  limits,  and  the  normal  value  for  characters 
which  have  been  arranged  in  this  manner  will  then  lie  within  the 
limits  of  the  group  which  contains  the  greatest  number  of  individual 
values. 

In  Tables  12-15,  inclusive,  certain  of  the  bacterial  ratios  have 
been  grouped  in  this  manner,  the  size  of  each  group  being  expressed 
as  the  percentage  which  the  number  of  samples  included  in  the  group 
is  of  the  total  number  of  samples  examined.  The  various  50°  ratios 
have  been  omitted,  for  the  reason  that  a  fair  normal  value  cannot 
be  obtained  by  the  group  method  when  the  investigation  covers 
less  than  25  samples.  On  the  other  hand,  determinations  of  bac- 
teria and  B.  coli  in  Merrimack  River  water  have  been  made  on 
thousands  of  samples,  while  on  certain  other  classes  of  samples 
many  hundred  determinations  have  been  made,  with  the  result 
that  the  normal  ratios  for  such  waters  are  correspondingly  accu- 
rate. 


Bacteria  Developing  at  Different  Temperatures     245 

The  normal  ratio  between  the  20°  and  jo°  bacteria  for  the  Merri- 
mack River  samples  appears  to  be  about  5,  40  per  cent  of  the  samples 
having  ratios  between  i  and  5,  9  per  cent  being  below  i,  and  the 
remaining  51  per  cent  being  well  distributed  above  5.  As  the  waters 
become  purer,  the  ratios  become  more  varied.  The  normal  limit  for 
Applied  216  is  still  between  i  and  10,  but  is  apparently  somewhat 
higher  than  in  the  case  of  the  river  water.  The  normal  limit  for 
the  effluent  from  Filter  No.  216,  like  the  river  water,  is  between 
I  and  5,  but  the  majority  of  the  other  ratios  fall  below  rather  than 
above  those  limits.  The  purer  effluents  from  the  water  filters  oper- 
ating at  low  rates  are  characterized  by  the  large  percentage  of  samples 
having  ratios  of  o,  and  by  the  distribution  of  the  remaining  samples 
among  the  higher  ratios,  which  fact  is  still  further  emphasized  when 
we  come  to  the  pond  waters. 

The  normal  ratio  between  the  20°  and  40°  bacteria  again  falls  be- 
tween I  and  5  for  samples  from  the  Merrimack  River  and  from  Ap- 
plied 216.  In  the  same  group  must  be  included  the  effluent  from 
mechanical  Filter  No.  216.  The  ratios  for  the  other  water  filters  are 
more  widely  distributed,  19  per  cent  of  the  samples  not  showing  any 
growth  at  40°,  and  37  per  cent  having  ratios  above  15.     The  group 


TABLE  12. 

Distribution  of   the   Ratios   between    20°    Bacteria   and   Bacteria   at   30°   and   40°    among 
Different  Samples  for  Various  Classes  of  Waters. 


Per  Cent  of  Sauples  Having  Ratios 


Of  o 


Less  than 

I 


Between 
I  and  5 


Between 

5  and  10 


Between 
10  and  15 


Above  :s 


for    ratios   between    20      AND    30      BACTERIA. 


Merrimack  River  . 

Applied  216 

Filter  No.  216 

Other  water  filters . 
Fends 


2 

7 

40 

JS 

13 

0 

10 

31 

IP 

13 

24 

8 

40 

16 

8 

41 

0 

9 

10 

0 

77 

0 

3 

5 

5 

23 

10 

4 
22 
10 


FOR    RATIOS   BETWEEN    20     AND   40      BACTERIA. 


Merrimack  River. . 

Applied  216 

Filter  No.  216 

Other  water  filters. 

Sea  waters 

Ponds 

Wells 


0 

IS 

63 

17 

0 

0 

15 

54 

19 

12 

12 

0 

S6 

34 

4 

19 

0 

3 

10 

22 

10 

0 

20 

33 

33 

40 

0 

3 

15 

10 

42 

18 

20 

0 

4 

5 
o 
4 
37 
6 

33 

7 


246 


Stephen  DeM.  Gage 


of  sea  waters,  containing,  as  it  does,  samples  probably  not  exposed 
to  pollution,  and  samples  which  are  grossly  polluted,  shows  a  more 
uniform  distribution  of  ratios  than  any  of  the  other  classes  of  waters, 
although  the  normal  ratio  for  samples  containing  bacteria  which  are 
able  to  grow  at  40°  appears  to  lie  between  5  and  10.  The  distinc- 
tion between  the  pond  waters  and  the  well  waters  is  quite  marked 
Forty-nine  per  cent  of  the  pond  samples  and  42  per  cent  of  the 
well  samples  did  not  contain  any  bacteria  growing  at  40°  C.  Of 
the  samples  which  did  contain  bacteria  of  this  type,  nearly  all  of  the 
pond  samples  have  high  ratios,  while  the  ratios  for  the  well  samples 
are  usually  very  low.  The  percentage  distribution  of  the  different 
ratios  between  the  20°  bacteria  and  the  bacteria  determined  at  30° 
and  40°  for  different  classes  of  waters  is  shown  in  Table  12. 

The  normal  ratios  between  the  20°  bacteria  and  the  jo°  acid-pro- 
ducing organisms  follow  much  the  same  rule  as  the  20°-30°  bacteria 
ratios,  the  distinction  between  the  different  classes  of  water,  however, 
being  much  more  sharply  marked.  The  normal  ratio  for  Merri- 
mack River  water  lies  between  i  and  5,  and  with  a  large  majority 
of  the  ratios  above  5,  falling  below  10.  With  the  Applied  216,  while 
the  normal  ratio  lies  between  the  same  limits  as  for  the  river  water, 
the  other  ratios  are  about  evenly  distributed  above  and  below  those 
limits.  Again,  with  the  effluent  from  Filter  No.  216  the  normal 
ratio  is  between  5  and  10,  but  the  number  of  samples  having  ratios 

TABLE  13. 

Distribution   of    Ratios    Between    20°    Bacteria    and    30°    Acid-Producing    Bacteria    among 
Different  Samples  for  Various  Classes  of  Waters. 


Per  Cent  of  Samples  Having  Ratios 

Of  0 

Less  than 

I 

Between 

I  and  5 

Between 
5  and  10 

Between 
10  and  15 

Above  IS 

Merrimack  River 

0 

8 

36 

66 

90 

10 

23 

12 

0 

0 

so 

42 

44 

6 

3 

29 

23 

4 

13 

7 

8 
4 
4 
6 

0 

3 
0 
0 
9 

0 

Applied  216 

Filter  No.  216 

Other  water  filters 

Ponds 

above  5  is  small,  and  a  considerable  percentage  of  the  samples  did 
not  show  acid-producing  bacteria.  As  we  pass  to  the  effluents  from  the 
slow  water  filters,  and  thence  to  the  pond  waters,  the  percentage  of 
samples  free  from  acid-producing  bacteria  increases,  while  the  normal 


Bacteria  Developing  at  Different  Temperatures     247 

ratios  for  such  samples  as  do  contain  bacteria  of  this  tyj)c  arc  alxive  5. 
The  figures  are  shown  in  Table  13. 

Normal  bacteria-B.  coli  ratios. — In  determining  the  normal  ratio 
between  the  20°  bacteria  and  the  B.  coli,  our  available  data  cover 
a  wider  variety  of  sources  and  a  larger  number  of  samples  than  is 
the  case  of  the  ratios  previously  discussed.  With  raw  sewages 
57  per  cent  of  the  samples  examined  have  ratios  between  i  and  5, 
and  26  per  cent  of  the  samples  have  ratios  between  5  and  10,  the 
other  ratios  being  distributed  on  both  sides  of  these  limits.  The 
normal  ratio  for  raw  sewages,  then,  is  probably  just  below  5. 
The  treatment  of  sewage  by  septic  action  or  by  straining  tends  to 
eliminate  abnormal  ratios,  as  is  shown  by  the  fact  that  75  per  cent 
of  the  septic  sewage  samples  and  83  per  cent  of  the  strained  sewage 
samples  have  ratios  between  the  i  and  5.  A  somewhat  smaller 
percentage  of  the  ratios  for  the  effluents  from  sewage  filters  is  found 
between  i  and  5,  although  the  normal  ratios  for  these  classes  of 
samples  still  remain  within  those  limits.  A  distinction  between 
the  effluents  of  the  sand  and  trickling  filters,  whose  action  is  entirely 
aerobic,  and  the  contact  filters,  whose  action  is  partly  anaerobic, 
is  manifest  by  the  considerable  percentage  of  samples  of  the  former 
classes  which  have  ratios  less  than  i,  as  compared  with  the  small 
percentage  of  the  latter  class.  The  distribution  of  the  ratios 
over  a  broader  scale  is  also  notable,  indicating  the  fluctuating 
character  of  the  types  of  bacteria  contained  in  the  effluents  from  all 
types  of  sewage  filters.  The  normal  ratios  for  the  Merrimack  River 
water  and  Applied  216  are  also  between  i  and  5,  with  a  large  major- 
ity of  the  ratios  outside  these  limits,  falling  below  i.  The  effluent 
from  Filter  No.  216  has  a  normal  ratio  between  i  and  5,  with  20 
per  cent  of  the  samples  having  a  ratio  of  o,  while  the  effluents  from 
the  slow  water  filters  are  characterized  by  the  fact  that  50  per  cent 
of  the  samples  have  a  ratio  of  o,  the  other  ratios  being  fairly  w.-ll 
distributed,  with  a  normal  ratio  somewhat  above  5.  The  variable 
character  of  the  sea-water  samples  is  evident  from  the  distribution 
of  the  ratios.  With  this  class  of  samples  the  normal  ratio  again  falls 
between  i  and  5,  39  per  cent  of  the  samples  having  ratios  Ix^wecn 
those  limits,  with  21  per  cent  lying  between  5  and  10,  14  jkt  cent 
falling  below  i,  and  19  per  cent  with  a  ratio  of  o.     The  ditTcrcncc 


248 


Stephen  DeM.  Gage 


between  the  pond  and  well  waters  and  the  other  classes  of  samples 
is  indicated  by  the  large  number  of  samples  which  do  not  contain 
acid-producing  bacteria  at  40°  C.  A  distinction  occurs  between 
the  pond  and  well  waters,  as  shown  by  the  fact  that  the  ratios  for 
the  former  are  distributed  among  the  higher  values,  while  the  ratios 
for  the  latter  are  distributed  among  the  lower  values.  The  normal 
ratio  is  not  apparent  for  the  pond  waters,  but  the  normal  ratio  for 
such  samples  of  well  water  as  contained  bacteria  of  this  type  falls 
between  the  limits  previously  noted  for  other  classes  of  samples; 
that  is,  between  i  and  5.  The  distribution  of  the  bacteria-5.  coli 
ratios  for  all  samples  from  these  13  different  sources  is  shown  in 
Table  14: 

TABLE  14. 

Distribution  of  Bactekia-B.  coli  Ratios  Among  Different  Samples   for   Various    Classes   of 

Waters. 


Above  15 


Lawrence  sewage 

Septic  sewage 

Strained  sewage 

Sand  filter  effluents..  .  . 
Contact  "  "  ..  .  . 
Trickling  filter  efi3uents 

Merrimack  River 

Applied  216 

Filter  No.  216 

Other  water  filters 

Sea  waters 

Pond  waters 

Wells 


Per  Cent  of  Samples  Having 

Ratios 

Ofo 

Less  than 

Between 

Between 

Between 

I 

I  and  5 

5  and  10 

10  and  IS 

0 

6 

57 

26 

8 

0 

10 

75 

10 

S 

0 

17 

83 

0 

0 

0 

31 

41 

II 

5 

0 

5 

48 

22 

13 

0 

26 

44 

II 

11 

0 

36 

60 

2 

2 

4 

27 

58 

11 

0 

20 

0 

68 

8 

4 

50 

3 

13 

19 

6 

19 

14 

39 

21 

2 

82 

0 

3 

5 

3 

82 

.S 

12 

I 

0 

3 
o 
o 

12 

12 

8 

o 
o 
o 
9 
5 
7 
o 


In  Table  15  is  shown  the  distribution  of  the  ratios  between  the 
bacteria  and  the  acid-producers  developing  at  20°,  30°,  and  40°  C. 
In  computing  the  values  in  this  table  only  samples  are  included 
which  contained  bacteria  capable  of  development  at  the  given  tem- 
perature. The  difference  between  these  ratios  and  the  ones  pre- 
viously discussed  consist  in  their  much  wider  distribution,  and  this 
has  made  necessary  the  grouping  of  the  ratios  between  somewhat  wider 
limits. 

The  normal  ratios  between  the  bacteria  and  acid-producers  at  20° 
for  Merrimack  River  water  and  Applied  216  appear  to  be  about 
20  in  each  case.     The  normal  ratio  for  the  effluent  from  Filter  No. 


Bacteria  Developing  at  Different  Temperatures     249 

216  is  well  below  20,  probably  about  10;  while  the  ratio  for  other 
filtered  waters  appears  to  be  somewhat  higher,  lying  between  15 
and  20.  The  pond  waters  are  characterized  by  very  low  ratios, 
the  normal  ratio  being  about  i,  41  per  cent  of  the  samples  having 
ratios  below  i,  and  31  per  cent  between  i  and  20. 

The  ratios  between  the  bacteria  and  a^id- producers  at  jo°  are  higher 
than  the  corresponding  ratios  at  20°.  The  normal  ratios  for  the 
river  water  and  Applied  216  lie  between  40  and  80,  probably  being 
about  60  in  each  case.  A  majority  of  the  samples  from  Filter  No. 
216  had  ratios  above  80,  with  32  per  cent  of  the  samples  having 
ratios  between  20  and  40,  the  remainder  of  the  samples  being  divi- 
ded in  two  equal  groups,  having  ratios  between  i  and  20,  and  between 
40  and  60.  With  the  effluents  from  the  slow  sand  filters  we  again 
find  a  majority  of  the  samples  having  ratios  above  80,  with  36  per 
cent  of  the  samples  evenly  divided  into  two  groups,  with  ratios 
between  20  and  40,  and  between  40  and  60,  respectively.  Seventy- 
five  per  cent  of  the  pond-water  samples  have  ratios  above  80,  while 
the  remaining  25  per  cent  have  ratios  between  40  and  60.  A  dis- 
tinction between  the  different  classes  of  water  is  seen  in  the  number 
of  samples  having  ratios  above  80,  about  25  per  cent  of  the  pol- 
luted water  samples,  50  per  cent  of  the  filtered  waters,  and  75  per 
cent  of  the  pond  waters  being  so  characterized. 

Normal  ratios  between  40°  bacteria  and  acid- producers. — In  study- 
ing the  ratios  between  the  bacteria  and  acid-producers  at  40°  we 
have  available  a  large  variety  of  sources  and  more  samples  from 
each  source,  so  that  our  results  are  more  conclusive.  The  normal 
ratio  for  the  river  water  appears  to  lie  just  above  60,  while  that  for 
Applied  216  appears  to  be  between  70  and  80.  Over  55  per  cent 
of  the  Filter  No.  216  samples  have  ratios  above  80,  thus  placing 
the  normal  ratio  for  this  water  just  above  80.  With  the  effluents 
from  the  slow  water  filters  we  find  the  ratios  more  uniformly  distrib- 
uted, and  although  the  normal  ratio  appears  to  be  about  60,  its 
location  is  not  distinctively  marked.  The  pond  water  normal  ratio 
is  located  between  70  and  80,  and  the  ratios  are  distributed  less  widely 
all  being  above  20,  as  was  the  case  with  the  samples  from  Applied 
216  and  Filter  No.  216.  The  sea  waters  are  again  divided  into  two 
groups,  19  per  cent  of  the  samples  having  ratios  less  than  i,  while 


250 


Stephen  DeM.  Gage 


nearly  all  the  remaining  samples  had  ratios  above  40.  The  normal 
ratio  for  this  latter  group  of  sea-water  samples  is  probably  between 
50  and  60.  The  well-water  samples  are  characterized  by  the  fact 
that  68  per  cent  of  the  samples  have  ratios  less  than  i,  the  ratios 
for  the  remaining  samples  being  distributed  in  a  fairly  uniform 
manner. 

TABLE   15. 

Distribution  of  the  Ratios  between  the  Total  Bacteria  and  the  Acid-Producing  Bacteria 
at  20°,  30°,  and  40^  among  different  samples  for  various  classes  of  waters. 


Per  Cent  of  Samples  having  Ratios 


Less  than 


Between  i 
and  20 


Between  20 
and  40 


Between  40 
and  60 


Between  60 
and  80 


Above  80 


PLATES  incubated   AT   20     C. 


Merrimack  River  . 

Applied  216 

Filter  No.  216  . .  .  . 
Other  water  filters. 
Ponds  

Merrimack  River  . 

Applied  216 

Filter  No.  216  .  .  .  . 
Other  water  filters 
Ponds  

Merrimack  Ris'er.  . 

Applied  216 

Filter  No.  2 16  . . .  . 
Other  water  filters. 

Ponds 

Sea  waters 

WeUs    


10 

38 

40 

10 

0 

12 

31 

31 

19 

7 

12 

60 

16 

4 

0 

19 

31 

41 

0 

9 

45 

31 

13 

3 

3 

PLATES  INCUBATED  AT  30     C. 


0 

8 

13 

28 

28 

0 

5 

16 

25 

29 

0 

6 

32 

6 

0 

0 

9 

18 

18 

0 

0 

0 

0 

25 

0 

23 
25 

S6 
55 
75 


PLATES  INCUBATED  .AT  40     C. 


0 

5 

13 

23 

42 

0 

0 

16 

20 

32 

0 

0 

10 

10 

25 

0 

12 

13 

25 

31 

0 

0 

14 

14 

29 

19 

I 

2 

48 

18 

68 

2 

4 

II 

4 

17 
32 

55 
19 
43 
12 
II 


Causes  of  variation  in  bacterial  contents  0}  Merrimack  River  water. — 
As  fluctuating  factors  which  are  most  liable  to  influence  the  numbers 
of  bacteria  and  B.  coli,  and  the  ratio  between  bacteria  and  B.  coli, 
in  the  water  from  such  a  source  as  the  Merrimack  River,  we  have 
the  volume  of  water  flowing  in  the  river,  the  temperature  of  the  water, 
and  the  amount  of  dissolved  oxygen  in  the  water.  For  a  clear  under- 
standing of  the  influence  of  these  three  factors  it  is  first  necessary 
to  consider  the  influence  of  the  various  factors  upon  one  another. 
It  is  quite  generally  understood  that  the  amount  of  oxygen  dissolved 
in  water  varies  inversely  as  the  temperature,  a  greater  amount  of 


Bacteria  Developing  at  Different  Temperatires     2si 


oxygen  being  present  in  the  winter,  when  the  water  is  cold,  than 
in  the  summer,  when  the  water  is  warm.  The  relationshij)  between 
these  two  factors  is  shown  quite  clearly  in  Table  i6.  A  study  of 
the  temperatures  of  the  water,  and  of  the  amounts  of  dissolved  oxygen 
at  times  of  high  and  low  water,  reveals  the  fact  that  the  average 
temperature  was  high,  and  the  average  amount  of  dissolved  oxygen 
was  low,  when  the  river  was  low,  these  factors  decreasing  and 
increasing  respectively  as  the  volume  of  flow  in  the  river  increased. 
The  average  results  of  eight  years'  determinations  of  the  tempera- 
ture and  dissolved  oxygen,  arranged  according  to  the  volume  of 
water  flowing  in  the  river,  are  shown  as  follows  in  Table  i6 : 


table  i6. 
Relation  ofTemperatureandDissolvedOxycen  to  Volumeof  Merrimack  River. 

Flow  of  River — Cubic  Feet  per  Square  Mile  of 
Watershed 

Temperature 
Water 

of 

Dissolved  Oxygen 

— Parts  per 

100,000 

I>ess  than  i 

Between  i  and  2 

60 
48 

47 
46 

073 
I  00 

'*       2  and  4 

Above  4 

III 
'    13 

The  effect  of  different  amounts  of  dissolved  oxygen  in  the  water 
is  shown  quite  clearly  in  Table  17,  in  which  the  bacterial  results 
have  been  arranged  according  to  the  amount  of  oxygen  present.  The 
numbers  of  bacteria  and  of  B.  coli  were  both  at  a  maximum  when 
the  dissolved  oxygen  in  the  water  was  between  0.50  and  0.75  parts 
per  100,000.  As  the  amount  of  oxygen  increased  or  decreased 
from  these  limits,  the  numbers  of  bacteria  and  B.  coli  decreased, 
the  numbers  of  B.  coli  decreasing  much  more  rapidly  with  the  increase 

table   17. 

Relation  between  Amount  of  Dissolved  Oxygen  and  the  Bacterial  Contents  of   MF.RRruAcc 

River  Water. 


Dissolved  Oxygen-Parts  per   100,000 

Less  than  o .  so 

Between  o . 50  and  0.75 

"      0.7s     "     1.00 

"       1. 00      "      I.2S 

More  than  125 


Bacteria  per  c.c. 


6,200 
10,000 
6,000 
4,200 
5,200 


B.  coli  per  c.c. 


74 

t04 

47 

37 


Bactfrio-fl.  eoti 
Ratio 


t  42 
1.16 

o  87 
o  (W 
o   59 


in  dissolved  o.xygen  than  did  the  bacteria.     This  is  shown  by  the 
bacteria-5.  coli  ratios  in  the  last  column  of  the  table,    the    ratios 


252 


Stephen  DeM.  Gage 


decreasing  in  proportion  as  the  amount  of  oxygen  in  the  water  in- 
creased. 

The  seasonal  distribution  of  B.  coli  in  samples  of  Merrimack 
River  water  has  been  discussed  in  a  former  pubhcation'^  in  which 
it  was  shown  that  the  average  numbers  of  B.  coli  were  higher  during 
the  summer  months  than  during  the  winter  months.  A  similar 
variation  can  be  noted  in  Table  i8,  in  which  the  bacteria  and  B. 
coli  and  the  ratio  between  the  two  have  been  arranged  according 
to  the  temperature  of  the  water  at  the  time  the  samples  were  col- 
lected. The  maximum  numbers  of  both  bacteria  and  B.  coli  occurred 
in  samples  from  both  locations  when  the  temperature  of  the  water 
was  between  60°  and  70°  F.,  and  the  minimum  numbers  of  bacteria 
were  found  when  the  temperature  was  between  40°  and  50°  F.  The 
temperatures  at  which  the  minimum  numbers  of  B.  coli  were  found 
in  samples  from  the  two  locations  do  not  agree,  being  lowest  in  the 
canal  water  when  the  temperature  was  between  40°  and  50°  F.,  and 
lowest  in  the  Intake  samples  when  the  temperature  was  between 
30°  and  40°  F.  The  bacteria-jB.  coli  ratios  were  highest  when  the 
temperature  was  highest  and  lowest  when  the  temperature  was 
lowest,  the  values  for  intermediate  temperatures,  however,  appearing 
to  follow  no  definite  curve. 

TABLE  18. 
Relation  between  Temperature  and  Bacterial  Contents  of  Merrimack  River  Water. 


Temperature  of  Water — 
Degrees  F. 

Bacteria  per  c.c. 

B.  coli   PER  CO. 

Bacteria-B.  coli 
Ratio 

Canal 

Intake 

Canal 

Intake 

Canal 

Intake 

Below  40° 

Between  40°  and  50° 

50°  and  60° 

5.800 
4,000 
6,400 
11,300 
S.200 

8,800 
4.S00 
9,500 
15,300 
6,700 

43 

36 

44 

100 

70 

58 

58 

no 

82 

0.83 
001 
0.88 
1.08 

I    39 

0.72 
1 .  10 
0  99 
0.97 
I. 21 

"       6o°aiid7o° 

Above  70° 

The  averages  of  bacterial  determinations  and  the  bacteria-.B. 
coli  ratios,  arranged  according  to  the  volume  of  water  flowing  in 
the  river,  are  shown  in  Table  19.  Both  bacteria  and  B.  coli  decreased 
as  the  volume  of  the  river  increased.  We  should  expect  this  to  be 
the  case  in  a  river  such  as  the  Merrimack,  in  which  a  large  proportion 
of  bacteria  and  B.  coli  are  contributed  by  the  sewage  entering  that 
river,  the  effect  of  dilution  overbalancing  other  factors,  such  as  wash- 


Bacteria  Developing  at  Different  Temperatures     253 

ings  from  cultivated  fields,  etc.,  when  averages  of  a  large  numijcr 
of  samples  extending  over  a  considerable  period  are  included.  The 
ratios  between  the  bacteria  and  B.  coli  on  samj)k's  from  the  two  sources 
do  not  agree,  the  highest  ratios  being  obtained  for  the  canal  samples 
when  the  river  was  low,  and  the  lowest  ratios  when  the  river  was 
high.  With  the  Intake  samples,  however,  the  lowest  ratios  occurred 
at  a  time  of  medium  high  water,  and  the  highest  at  extreme  high 
water. 

TABLE   19. 
Relation  BETWEEN  Volume  OF  Flow  AND  the  Bacterial  Content  op  Merrimack  River  Water. 


Flow  of  River,  Cubic  Feet 
PER    Second    per    Square 

Bacteria  per  c.c. 

B.  coli  PER  c.c. 

BACTERIA-iU.  coli 

Ratio 

Mile  of  Watershed 

Canal 

Intake 

Canal 

Intake 

Canal 

Intake 

Less  than  i   . 

7.500 
6,800 
3.600 
3.400 

10,800 
6,200 
5.600 
3,100 

66 
SO 
20 
16 

88 
51 
30 
29 

1.07 

°  P 
0  83 

0.63 

0  97 
0  99 
0  56 
1.07 

Between  i  and  2 

"           7   and  ^^ 

Above  4 

CONCLUSIONS. 

The  apparent  discrepancies  which  have  occurred  between  the 
results  of  bacteriological  and  of  chemical  analysis  of  water  have 
caused  a  reasonable  doubt  in  the  minds  of  many  persons,  having 
occasion  to  use  such  results,  as  to  the  practical  value  of  the  bacterio- 
logical determinations.  That  such  discrepancies  do  exist  cannot 
be  denied,  and  that  the  bacteriological  results  instead  of  the  chemical 
results  should  be  doubted  is  natural,  considering  that  the  complete 
chemical  analysis  is  composed  of  a  number  of  individual  factors, 
each  of  which  has  received  long  and  careful  study,  while  the  bacterio- 
logical procedure  is  confined  to  a  determination  of  the  numbers  of 
bacteria,  and  of  the  presence  or  absence  of  one  specific  type  of  bac- 
teria, i.  e.,  the  colon  type.  If  the  chemical  analysis  of  water  were 
confined  to  a  determination  of  the  total  nitrogen  content,  instead 
of  dividing  that  nitrogen  content  into  its  constituent  parts — free 
ammonia,  albuminoid  ammonia,  nitrates,  and  nitrites — and  only  a 
qualitative  test  were  made  for  chlorine,  the  interpretation  of  the  char- 
acter of  the  water  from  the  chemical  results  would  be  as  frequently 
in  error  as  when  a  similar  interpretation  based  on  the  usual  bacterio- 
logical results  is  attempted.     If,  on  the  other  hand,  complete  and 


254  Stephen  DeM.  Gage 

varied  data  regarding  the  bacterial  contents  of  a  water  could  be 
obtained,  the  apparent  discrepancies  would  cease  to  exist,  and  the 
chemical  and  bacteriological  analyses  would  supplement  and  con- 
firm one  another,  rendering  the  correct  interpretation  of  the  quality 
of  the  water  a  comparatively  simple  matter.  The  object  of  the  present 
paper  has  been  to  supply  a  portion  of  the  information  necessary  for  the 
etablishment  of  bacteriological  procedures  by  which  a  more  thorough 
knowledge  of  the  bacterial  content  of  the  water  may  be  obtained. 
Nearly  all  of  the  information  desired  concerning  the  bacterial  content 
of  water  may  be  obtained  by  the  use  of  selective  media,  by  the  use 
of  selective  temperatures,  or  by  a  proper  combination  of  the  two. 
In  the  present  investigation  the  selective  action  of  four  different 
temperatures,  20°,  30°,  40°,  and  50°  C,  and  two  different  media, 
regular  agar,  and  litmus-lactose  agar,  in  determining  the  bacterio- 
logical contents  of  a  number  of  different  kinds  of  water,  have  been 
studied;  and  while  the  results  obtained  have  been  in  many  cases 
inconclusive,  and  in  other  cases  too  few  in  number  to  warrant  the 
drawing  of  any  far-reaching  conclusions,  they  indicate  in  a  measure 
the  procedures  which  must  be  followed  in  order  to  place  the  bacterio- 
logical analysis  of  water  on  the  same  plane  as  the  chemical  analysis. 

The  results  of  the  investigation  may  be  summarized  as  follows: 
The  numbers  of  bacteria  determinable  upon  agar  or  gelatin  are 
very  closely  approximated  by  the  numbers  determined  upon  litmus- 
lactose  agar,  while  the  substitution  of  the  latter  medium  for  the 
former  allows  of  the  simultaneous  determination  of  the  total  bacteria 
and  of  the  acid-producing  bacteria  without  appreciably  increasing  the 
labor  involved  in  the  determination.  The  numbers  of  the  two  classes 
of  bacteria  so  determined  indicate  more  completely  the  character  of 
the  water  than  would  the  numbers  of  either  class  determined  alone. 

It  is,  of  course,  unnecessary  to  discuss  the  significance  of  the 
numbers  of  bacteria  determined  at  20"  C.  The  number  of  acid- 
producing  organisms  determined  at  20°  C,  however,  is  an  important 
check  upon  the  total  numbers.  In  one  case  we  saw  that  with  two 
well  waters,  one  polluted  and  one  not  polluted,  the  numbers  of  bac- 
teria in  the  pure  well  water  were  about  twice  as  great  as  in  the  pol- 
luted water,  but  the  numbers  of  acid-producing  bacteria  showed 
the  high  numbers  for  the  pure  water  to  be  misleading. 


Bacteria  Developing  at  Different  Temperatures     255 

The  numbers  oi  bacteria  and  of  acid-producing  organisms  deter- 
mined on  litmus-lactose  agar  at  30^  C.  after  24  hours'  incubation 
are  smaller  than  the  numbjrs  determined  at  20°  C.  after  two  to  four 
days'  incubation;  but  even  with  these  smaller  numbers  the  distinction 
between  the  polluted  waters  and  the  waters  of  good  (juality  is  more 
sharply  marked  than  is  the  case  with  the  numbers  determined  at  the 
lower  temperatures.  Determinations  at  this  temperature  appear  to 
be  especially  applicable  to  the  control  of  water  fillers,  since  the 
relative  purity  of  the  raw  and  filtered  waters,  and  the  expression  of 
the  hygienic  efficiency  of  such  filters  as  the  percentage  removal 
of  bacteria,  are  practically  identical  with  similar  determinations 
made  at  20°,  while  the  advantage  obtained  by  having  the  results 
available  within  24  hours  would  prove  invaluable  in  controlling  the 
operations  of  the  filters  and  preventing  any  serious  change  in  the 
character  of  the  filtered  water. 

The  numbers  of  bacteria  determined  at  40°  C.  are  of  great  inter- 
est, since  in  this  class  of  bacteria  must  be  included  the  disease-pro- 
ducing organisms.  The  distinction  between  waters  of  different 
kinds  and  between  waters  of  the  same  kind  representing  difTerent 
degrees  of  pollution  is  well  marked  by  counts  at  this  temperature. 
The  significance  of  the  numbers  of  acid-producing  bacteria  deter- 
mined at  40°  C— i.  e.,  bacteria  of  the  colon  type — is  well  known. 
It  may  be  said,  however,  that  the  usual  practice  of  making  qualita- 
tive determinations  of  the  presence  or  absence  of  B.  colt  should  be 
supplemented  by  quantitative  determinations.  It  is  the  belief  of  the 
writer,  based  on  experience  gained  in  the  present  study,  that  con- 
siderable numbers  of  bacteria  of  the  colon  type  may  occur  in  waters 
which  are  supposedly  quite  pure,  judging  from  their  tt)tal  bacterial 
content,  while  on  other  occasions  a  positive  test  for  B.  coli  may  be 
caused  by  an  isolated  organism  of  that  type.  In  the  one  case  the 
water  would  be  open  to  suspicion,  in  the  other  case  it  would  prob- 
bably  be  relatively  harmless,  although  no  such  distinction  could 
be  made  from  the  results  of  the  qualitative  tests. 

The  results  of  determinations  of  bacteria  and  of  acid-producing 
organisms  which  are  able  to  develop  at  a  temperature  of  50°  C. 
are  quite  interesting.  We  see  that  relatively  large  numbers  of  bac- 
teria of  this  type  occur  in  sewages  and  in  the  effluents  from  sewage 


256  Stephen  DeM.  Gage 

filters  in  which  the  free  passage  of  the  sewage  is  practically  unre- 
strained, while  they  are  entirely  absent  from  surface  and  ground 
waters  which  have  not  been  exposed  to  any  considerable  pollution. 
It  is  rather  surprising,  however,  that  the  numbers  of  bacteria  of 
this  type  in  very  polluted  Merrimack  River  water  were  not  larger. 

The  information  to  be  obtained  by  counts  of  bacteria  and  acid- 
producing  organisms  at  any  one  of  the  above  temperatures  is  greatly 
increased  by  the  combination  of  the  results  obtained  from  counts 
at  two  or  more  temperatures,  and  this  information,  is  much  more 
clearly  shown  if  we  express  the  relationship  between  the  counts 
at  the  different  temperatures  methematically.  Many  individual 
differences  between  different  waters  are  indicated  by  the  bacterial 
ratios  which  would  not  be  apparent  in  the  results  of  the  counts. 

The  writer  is  inclined  to  believe  that  a  combination  of  counts 
at  20°  C.  and  at  40°  C.  with  corresponding  ratios  will  yield  informa- 
tion which  will  enable  us  to  understand  many  of  the  hitherto  unex- 
plainable  discrepancies  in  the  results  of  bacterial  analysis  of  water. 
The  combination  of  20°  and  30°  counts  appears  to  be  of  less  value 
while  the  value  of  50°  counts  in  combination  with  the  other  counts 
has  not  been  sufficiently  studied  to  determine  their  applicability. 

In  conclusion,  the  writer  wishes  to  acknowledge  his  indebtedness 
to  the  various  members  of  the  laboratory  force  at  the  Lawrence 
Experiment  Station  for  assistance  in  making  the  many  determina- 
tions, and  to  Mr.  H.  W.  Clark,  chemist  in  charge  of  the  station, 
whose  hearty  co-operation  has  made  the  investigation  possible. 

REFERENCES. 

1.  Fuller.     Amer.  Pub.  Health  Assoc.  Rep.,  1895,  20,  p.  381. 

2.  Hesse  and  Nieder.     Ztschr.  /.  Hyg.,  1898,  29,  pp.  29,  454. 

3.  Whipple.     Tech.   Quarterly,   1902,   15,   p.    127. 

4.  G.  Hesse.     Ztschr.  f.   Hyg.,    1903,   44,   p.    i. 

5.  Gage  and  Phelps.     Amer.  Pub.  Health  Assoc.  Rep.,  1901,  27,  p.  392. 
Gage  and  Adams.     Jour.  Infect  Dis.,  1904,  i,  p.  358. 

6.  Report  of   Committee  on   Standard  Methods  of  Water   Analysis,   Jour.   Inject. 

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7.  WuRZ.     Arch,   de   med.    exp.,    1892,   4,    p.    85. 

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Bacteria  Developing  at  Different  Temperatures     257 

11.  MlQUEL.     Manuel  pratique  d^analyze  bacteriologique  des  eaux,  Paris,  1891;     I^s 

Organismes  vivants  de  Vatmosphhre,   Paris,    1893,   p.    182. 

12.  Macfadyen  and  Blaxall.  Jour.  Path,  and  Bact.,  1895,  3,  p.  87. 

13.  Rabinowitsch.     Zeitschr.    /.    Hyg.,    1895,    20,  p.  154. 

14.  Houston.     Sup.   28th  Ann.   Rep.,  Local  Gov.   Bd.,   London,    1898-99. 

15.  Clark  and  Gage.     J4th  Ann.  Rep.,  Mass.  Board  of  Health,  1902,  p.  250. 


THE  TOXIC  EFFECT  OF  CERTAIN  ACIDS  UPON  TYPHOID 

AND  COLON  BACILLI  IN  RELATION  TO  THE 

DEGREE  OF  THEIR   DISSOCIATION* 

C.-E.    A.    WiNSLOW    AND   E.    E.    LOCHRIDGE. 
(From  the  Biological  laboratories  of  the  Massachusetts  Institute  oj  Technology.) 

I.     INTRODUCTION. 

The  researches  of  the  physical  chemists,  under  the  leadership  of 
Arrhenius  and  Nernst,  have  shown  that  certain  substances  in  aqueous 
solution  become  dissociated  or  broken  up  into  electrically  charged 
part -molecules  (atoms  or  groups  of  atoms),  which  are  called  ions. 
The  extent  to  which  this  occurs  varies  with  different  substances  and  is 
greatest  in  the  most  dilute  solutions.  With  strong  acids  and  bases, 
and  their  salts,  it  is  practically  complete  at  a  strength  of  o.ooi  normal. 
With  such  solutions  it  is  evident  that  any  effect,  chemical  or  physio- 
logical, which  they  exert,  must  be  due  to  the  dissociated  ions.  The 
properties  of  a  dilute  solution  of  sodium  chloride  are  the  properties 
of  sodium  and  chlorine  ions,  and  the  properties  of  hydrochloric  acid, 
of  hydrogen  and  chlorine  ions.  By  the  comparison  of  a  series  of 
properly  selected  compounds  it  is  easy  to  determine  the  specific 
influence  of  each  ion.  The  study  of  the  toxic  action  of  various 
substances  in  the  light  of  these  facts  promises  to  be  of  great  assis- 
tance in  the  development  of  a  rational  theory  of  disinfection. 

The  first  definite  statement  of  the  relation  between  dissociation 
and  disinfectant  power  with  which  we  are  familiar  was  made  by 
Dreser.  This  author  (Dreser,  1893),  in  a  study  of  the  pharmaco- 
logical value  of  various  salts  of  mercury,  found  that  the  double 
hyposulphite  of  mercury  and  potassium  was  much  less  poisonous 
than  other  compounds  containing  the  same  amount  of  mercury,  and 
explained  the  phenomenon  by  the  fact  that  this  salt  on  dissociation 
does  not  set  free  mercury  ions,  but  breaks  up  into  potassium  at  the 
cathode  and  Hg  S^O^  at  the  anode.  His  experiments  were  made  on 
yeast  cells,  frogs,  and  fishes.  In  the  former  case  he  found  it  possible 
to  prevent  all  development  in  a  yeast  culture  by  mercury  salts,  and 

♦Received  for   publication   March   5,    1906. 

258 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli        259 

then  by  the  addition  of  potassium  hyposulphite  to  permit  fermenta- 
tion without  precipitating  any  of  the  mercury,  simply  by  the 
formation  of  the  differently  dissociated  double  salt. 

Scheurlen  and  Spiro  (1897)  confirmed  the  conclusions  of  Dreser 
as  to  the  correlation  between  dissociation  and  disinfectant  action 
among  the  mercury  salts,  and  extended  them  to  cover  certain  com- 
pounds of  iron.  At  the  same  time  they  maintained  that  in  other 
cases  (the  ethylchlorid  and  ethylsulphate  of  mercury)  strong  disin- 
fectant action  was  apparently  due,  not  to  free  ions,  but  to  the 
undissociated  molecule. 

A  number  of  phenomena  which  had  long  been  empirically  familiar 
in  bacteriology  found  an  easy  explanation  on  the  electrolytic  theor}' 
of  disinfection.  The  effect  of  temperature  in  increasing  the  activity 
of  disinfectants,  for  example,  had  been  pointed  out  by  Koch  (18S1), 
and  later  by  Behring  (iSgo)  and  Hejder  (1892),  and  many  others. 
It  was  at  once  obvious  that  this  might  be  due  in  some  cases  to  the 
increased  dissociation  at  high  temperatures.  It  would  be  well  worth 
while  today  to  see  how  far  the  increased  activity  of  disinfectant  runs 
quantitatively  parallel  to  its  dissociation.  Again,  Minervini  (1898), 
and  other  investigators,  have  shown  that  various  antiseptic  agents 
(carbolic  acid,  chromic  acid,  mercuric  chloride,  and  silver  nitrate 
in  Minervini's  experiments)  are  much  less  active  in  alcoholic  than  in 
aqueous  solutions.  This  fact,  too,  is  easily  explicable  as  due  to  the 
diminished  dissociation  in  such  solvents. 

The  relation  between  dissociation  and  toxicity  was  put  upon  a 
sound  quantitative  basis  by  the  work  of  Kronig  and  Paul,  first  pub- 
lished in  1896  (Paul  and  Kronig,  1896),  and  in  fuller  detail  in 
the  next  year  (Kronig  and  Paul,  1897).  These  authors  carried  out 
an  elaborate  series  of  experiments  on  the  disinfectant  action  of  various 
salts,  bases,  and  acids  in  the  light  of  the  new  conclusions  of  physical 
chemistry.  The  details  of  the  investigation  were  arranged  with  the 
greatest  care  in  order  to  secure  comparable  results.  Spores  of  Bacillus 
anthracis  and  vegetative  cells  of  Micrococcus  aureus  were  used,  dried 
on  Bohemian  garnets.  By  using  a  definite  number  of  garnets  of  a 
certain  size  shaken  up  with  a  suspension  of  an  agar  culture,  after 
filtering  through  paper,  and  carefully  drying,  it  was  found  possible  to 
expose  approximately  the  same  number  of  cells  in  each  experiment. 


26o  C.-E.  A.  WiNSLOW   AND   E.  E.    LOCHRIDGE 

The  garnets  were  dried  for  12  hours  at  7°  and  exposed  in  platinum 
sieves  to  the  action  of  the  disinfectant  solution  studied.  The  tem- 
perature was  kept  constant  at  18°  C.  during  the  experiment,  and  after 
the  desired  time  had  elapsed  the  excess  of  disinfectant  was  carefully 
removed  by  appropriate  reagents  (neutralization  of  acids  and  bases, 
precipitation  of  heavy  metals  with  ammonium  sulphide,  etc.).  After 
thorough  rinsing,  the  garnets  were  shaken  up  with  water  to  detach 
the  cells,  which  were  then  plated  on  agar.  No  attempt  was  made 
accurately  to  fix  a  killing  point  by  testing  a  long  series  of  dilutions 
of  each  disinfectant,  and  no  exact  calculations  were  made  of  dissocia- 
tion. In  a  general  way,  however,  the  number  of  spores  of  B.  anthracis 
which  developed  after  treatment  for  a  given  time  varied  inversely 
with  the  amount  of  dissociation.  Thus  in  the  study  of  metallic  salts 
it  appeared  that  the  activity  of  various  compounds  of  mercury, 
silver,  copper,  and  gold  was  greatest  in  those  actively  dissociated, 
and  decreased  in  those  which  yield  less  free  metallic  ions.  Solutions 
of  mercuric  chloride  and  silver  nitrate,  in  alcohol,  where  no  dissocia- 
tion occurs,  showed  almost  no  disinfectant  action.  Furthermore, 
the  toxic  action  of  a  salt  having  poisonous  metallic  ions  was  markedly 
diminished  by  the  presence  of  a  non-toxic  salt  of  the  same  acid. 
This  is  in  accord  with  physico-chemical  theory;  in  any  solution  the 
ratio  between  the  undissociated  molecules  and  the  product  of  free 
anions  and  cations  is  constant,  so  that  the  addition  of  sodium  chloride 
to  mercuric  chloride  keeps  the  proportion  of  chlorine  ions  the  same, 
but  replaces  a  portion  of  the  mercury  ions  by  those  of  sodium.  So 
it  appeared  in  Kronig  and  Paul's  work  that  successive  additions 
of  sodium  chloride  to  mercuric  chloride  progressively  increased 
the  number  of  colonies  developing  after  the  usual  treatment.  In 
the  study  of  different  salts  of  the  same  metal  it  was  found  that  the 
acid  radical  may  also  exercise  considerable  influence  on  the  disinfect- 
ing power. 

With  bases  Kronig  and  Paul  found  the  same  general  relation  to 
hold,  ammonium  hydroxid,  which  is  weakly  dissociated,  being  a 
much  less  active  disinfectant  than  the  corresponding  compounds 
of  potassium,  sodium,  and  lithium.  The  authors  noted  in  a  general 
way  a  diminution  of  disinfectant  action  in  the  presence  of  organic 
compounds.     The  decrease  was  most  marked  with  the  halogens  and 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       261 

oxidizing  agents,  and  less  with  acids  and  bases.  Disinfectants  them- 
selves of  organic  nature  were  least  afifccted. 

The  particular  phase  of  the  subject  with  which  we  are  especially 
concerned,  the  disinfectant  action  of  the  acids,  was  not  exhaus- 
tively treated  in  this  investigation.  One  series  of  experiments  was 
made  with  normal  and  half-normal  solutions,  in  which  it  was  found 
that  hydrofluoric,  nitric,  and  trichloracetic  acids  in  normal  strength 
killed  all  the  anthrax  spores  in  120  minutes.  Normal  chloric,  hydro- 
bromic,  and  hydrochloric  acids  and  half-normal  oxalic  acid  left  a  few 
spores  alive  after  eight  hours.  Normal  sulphuric  acid  was  a  little 
less  effective,  and  normal  phosphoric,  formic,  and  acetic  acids  kft 
large  numbers  of  organisms  alive  after  eight  hours.  Hydrocyanic 
acid  in  normal  strength  showed  little  action  even  after  30  hours. 
The  investigators  conclude  that  there  is  a  general  relation  between  the 
action  of  the  acids  and  the  amount  of  dissociated  hydrogen  present ; 
but  there  appear  many  exceptions  to  a  strict  parallelism.  The 
authors  attribute  these  exceptional  effects  to  the  anion  or  the  undis- 
sociated  molecule,  and  point  out  that  in  more  dilute  solutions  they 
tend  to  disappear.  Thus,  0.06  normal  solutions  of  hydrochloric, 
chloric,  nitric,  and  trichloracetic  acids  showed  about  the  same 
disinfectant  action,  apparently  due  to  the  presence  of  an  approxi- 
mately equal  amount  of  dissociated  hydrogen. 

At  a  still  earlier  period  a  somewhat  similar  series  of  investigations 
to  those  of  Paul  and  Kronig  had  been  carried  out  in  another  field. 
This  was  a  study  of  the  relation  between  toxicity  and  dissociation 
as  measured  by  the  effect  of  various  salts  and  bases,  and  a  long 
series  of  organic  and  inorganic  acids,  on  the  higher  plants,  by 
Kahlenberg  and  True  (1896).  Their  method  consisted  in  determin- 
ing the  maximum  strength  of  solution  in  which  seedlings  of  Lupinus 
alhus  could  grow.  The  seedlings  were  exposed  for  15  to  24  hours, 
and  their  condition  determined  by  their  general  appearance  and  by 
the  growth  which  had  taken  place.  These  plants  proved  very 
sensitive  to  the  action  of  dilute  acid,  a  strength  of  from  0.00008  to 
0.00064  normal  killing  them  in  almost  ever)'  case.  It  is  interesting 
to  note  that  boric  acid  was  endured  in  10  times  this  strength.  In 
general,  the  poisonous  action  ran  parallel  with  tiie  degree  of  dis- 
sociation, but  certain  of  the  organic  acids  showed  relations  of  thiir 


262  C.-E.  A.  WiNSLOW   AND   E.  E.  LOCHRIDGE 

own.  The  authors  concluded  that  "in  the  case  of  plants  the  toxic 
action  of  solutions  of  electrolytes,  when  dissociation  is  practically 
complete,  is  due  to  the  action  of  the  ions  present.  When  dissocia- 
tion is  not  complete,  the  undissociated  part  of  the  electrolyte  may 
also  exert  a  toxic  effect."  Heald  (1896)  extended  the  work  of  Kahlen- 
berg  and  True  to  the  seedlings  of  three  other  flowering  plants,  and 
reached  the  same  general  conclusions.  All  these  authors  pointed 
out  clearly  that  the  effect  of  the  common  mineral  acids  is  due  to 
hydrogen  alone,  since  their  anions  have  almost  no  strong  toxic  action 
when  neutral  salts  are  used. 

The  next  work  along  these  general  lines  was  carried  out  in  another 
field  of  botany  by  Stevens  (1898),  at  the  University  of  Chicago,  the 
measure  of  viability  used  being  the  germination  of  fungus  spores.  A 
study  had  been  made  by  Wiithrich  (1892),  at  a  much  earlier  date  on 
the  toxicity  of  metallic  salts  and  acids  for  the  spores  of  fungi;  and 
Maillard  (1898  and  1899)  at  about  the  same  time  reported  experi- 
ments on  the  inhibition  of  the  growth  of  Penicillium  by  copper  salts. 
In  Stevens'  experiments  the  spores  were  inoculated  into  hanging-drop 
preparations  of  the  solutions  tested,  and  examined  for  development 
after  24  hours.  The  five  organisms  used  exhibited  marked  differ- 
ences in  their  susceptibility,  although  all  were  much  less  affected 
than  the  phanerogamous  seedlings,  requiring  a  strength  of  0.01-0.02 
normal  acid  to  inhibit  germination.  The  relative  toxic  effect  of 
various  substances  was  not  unlike  that  observed  by  Heald  and 
Kahlenberg  and  True.  Mercuric  chlorid  and  various  copper  salts 
proved  most  fatal,  the  acids  and  cyanides  being  less  active.  By 
the  comparison  of  various  substances  it  appeared  that  of  the  anions, 
CN,  CrO^,  Cr^O^,  and  OH  are  poisonous,  and  of  the  cations,  Hg, 
Cu,  and  H,  while  the  halogens  and  SO^  in  dilute  solutions  exert  no 
influence. 

A  still  more  exhaustive  study  of  the  effect  of  toxic  agents  upon 
the  fungi  was  made  by  Clark  in  the  next  year  (Clark,  1899).  This 
investigator  followed  the  same  general  method  as  that  of  Stevens, 
exposing  spores  in  hanging-drop  cultures  to  the  activity  of  the  agents 
to  be  tested.  The  cultures  were  divided  into  four  classes :  those  which 
grew  normally,  those  which  showed  irregular  or  retarded  growth, 
those  which  failed  to  develop  in  the  medium  tested,  but  grew  after 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       263 

transfer  to  fresh  beef  infusion,  and  those  which  entirely  succumbed 
to  the  action  of  the  toxic  substances.  The  wide  difference  Vx^tween 
the  concentration  of  acid  producing,  respectively,  injur)',  inhibition, 
and  death  was  one  of  the  most  interesting  results  of  these  experiments. 
As  in  Stevens'  work,  it  was  apparent  that  the  fungi  are  extremely 
resistant  to  disinfectants,  and  it  was  necessary  to  use  somewhat 
concentrated  solutions,  from  0.008  to  0.287  normal  acid,  for  killing. 
It  is  perhaps  partly  for  this  reason  that,  as  the  author  says,  "in  this 
study  no  new  evidence  has  been  adduced  supporting  the  theory  that 
the  chemical  activities  of  a  substance  are  due  wholly  or  chiefly  to  the 
ionized  portion."  On  the  other  hand,  it  was  held  that  "in  the  case 
of  several  acids,  ionization  lessens  the  chemical  activities  toward 
the  substances  involved  in  the  life-processes  of  the  plant."  This 
conclusion  is  based  on  calculations  of  the  specific  toxicity  of  each  ion 
and  molecule,  obtained  by  comparing  the  effects  of  different  com- 
pounds varying  from  each  other  in  one  element  or  group.  Thus,  hy- 
drochloric acid  in  the  solutions  used  was  over  90  per  cent  dissocia- 
ted and  since  experiments  with  the  similarly  dissociated  chloride  of 
potassium  showed  this  salt  to  be  practically  non-toxic,  it  is  evident  that 
its  action  was  due  to  the  combined  effect  of  the  hydrogen  ion  and  the 
undissociated  molecule.  Nitric  acid,  dissociated  in  the  same  proportion 
was  much  more  toxic.  Since  hydrogen  ions  are  equal  in  the  two  cases 
and  since  the  NO3  ion  is  harmless,  as  shown  by  experiments  with 
neutral  salts,  the  increased  effect  must  be  due  to  the  undissociated 
part  of  the  molecule  of  nitric  acid.  Clark  calculates  that  the  toxic 
value  of  one  molecule  of  undissociated  HNO3  is  7.7  times  that  of  an 
ion  of  hydrogen,  so  that  the  acid  actually  loses  nearly  seven-eighths 
of  its  disinfectant  power  by  becoming  ionized. 

The  effect  of  sulphuric  acid  was  about  the  same  as  that  of  hydro- 
chloric; since  it  is  less  dissociated,  the  author  attributes  an  appreciable 
influence  to  the  anion,  HSO4.  Acetic  acid,  at  the  strength  used,  is 
only  2  per  cent  dissociated,  so  that  its  high  toxic  effect  is  due  to  the 
un-ionized  molecule. 

The  results  obtained  in  this  series  of  exjRTimenis  with  hydro- 
cyanic acid  are  also  interesting.  Kronig  and  Paul  (i8q7)  found 
this  acid  almost  without  effect  on  anthrax  spores,  while  Kahlenlxrg 
and  True  (1896),  on  the  other  hand,  record  very  strong  toxic  action 


264  C.-E.  A.  WiNSLOW   AND   E.  E.  LOCHRIDGE 

on  the  seedlings  of  higher  plants.  In  Clark's  experiments  it  proved 
far  more  fatal  than  any  other  acid,  being  70  times  as  active  as 
hydrochloric.  The  molecule  at  the  concentrations  used  is  probably 
only  slightly  dissociated. 

In  some  ways  the  most  important  work  upon  this  subject  was 
the  very  careful  study  made  by  Bial,  of  the  antiseptic  action  of  the 
hydrogen  ion  of  dilute  acids  upon  yeast.  He  first  became  interested 
in  the  problem  from  a  consideration  of  the  causes  which  allow  pro- 
duction of  gas  in  the  stomach,  and  carried  out  his  earliest  experiments 
by  observing  the  gas  formation  in  yeast  cultures  in  the  presence  of 
various  substances  present  in  the  normal  gastric  juice.  This  series 
of  studies  (Bial,  1897)  showed  that  the  presence  of  albuminoid 
substances  or  of  sodium  chloride  effected  a  marked  restriction  of 
the  antiseptic  action  of  hydrochloric  acid.  Bial  at  this  time  did  not 
apply  physico-chemical  theories  to  the  explanation  of  these  phenom- 
ena; but  in  another  contribution  he  made  a  fuller  study  of  the 
problem.  His  later  experiments  (Bial,  1902)  were  again  made  with 
yeast  cells,  cultivated  in  fermentation  tubes  filled  with  grape-sugar 
solution  to  which  various  amounts  of  acid  had  been  added;  the 
antiseptic  action  was  inversely  registered  by  the  amount  of  gas 
produced.  The  advantage  of  this  method  is  its  great  delicacy; 
the  fermentative  power  of  the  yeast  responds  to  such  extremely 
minute  quantities  of  acid  that  the  ionic  effects  are  not  complicated 
by  other  actions  which  appear  in  stronger  solutions.  Bial  did  not 
make  exact  calculations  of  the  amount  of  dissociated  hydrogen 
necessary  to  inhibit  the  yeast,  but  he  found  that  a  general  relation 
existed  between  the  ionization  and  the  antiseptic  action.  The 
strongly  dissociated  acids  —  hydrochloric,  sulphuric,  nitric,  and 
trichloracetic — entirely  stopped  the  action  of  the  yeast  in  concentra- 
tions of  between  0.005  ^^d  0.008  normal.  Acids  of  an  intermediate 
grade — phosphoric,  formic,  and  oxalic — accomplished  the  same  effect 
when  o.oi  normal;  while  acids  still  less  dissociated — acetic,  benzoic, 
and  butyric — stopped  all  fermentation  only  when  0.04  to  0.07  normal. 
The  most  striking  feature  of  Bial's  work  was  a  series  of  experiments 
on  the  diminution  of  the  antiseptic  action  of  acids  by  the  addition 
of  neutral  salts  whose  action  is  to  decrease  the  dissociation  of  the 
acidic  hydrogen.     A  solution  of  o.oi   normal  formic  acid  and  0.3 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli        265 

normal  sodium  formate  showed  active  fermentation,  as  did  a  solution 
of  0,0166  normal  hydrochloric  acid  and  0.2  normal  sodium  chloride. 
The  same  phenomenon  was  observed  with  oxalates,  nitrates,  sulphates, 
and  acetates.  An  exhaustive  study  in  the  case  of  hydrochloric  acid 
showed  that,  while  a  certain  amount  of  sodium  chloride  diminished 
the  toxicity  of  the  acid,  a  much  larger  amount  actually  increased  it. 
Bial  attributes  this  to  a  catalytic  action  of  chlorine  ions,  but  it  seems 
to  us  that  the  facts  may  be  explained  more  simply  by  the  direct 
inhibiting  effect  of  the  sodium  chloride  and  its  ions.  Bial  found  that 
twice  normal  sodium  chloride  without  any  acid  prevented  fermenta- 
tion; and  it  is  quite  possible,  in  dealing  with  living  organisms,  that 
the  combined  effect  of  the  acid  and  the  chloride  would  be  inhibitory 
at  concentrations  which  with  either  acid  or  base  alone  might  allow 
fermentation  to  go  on.  Bial  studied  also  the  effect  of  hydrochloric 
acid  and  sodium  chloride  in  the  presence  of  peptone,  and  found  that 
the  yeast  would  bear  more  of  the  salt  than  in  the  presence  of  acid 
alone. 

The  experiments  of  Paul  and  Kronig  demonstrated  clearly  that 
in  certain  solutions  disinfectant  action  runs  parallel  with  the  presence 
of  dissociated  ions.  The  work  upon  the  higher  plants  and  the  mold 
fungi  confirmed  these  results,  but  showed  that  in  other  cases  the 
undissociated  molecule  is  of  great  importance.  Bial's  studies  brought 
out  clearly  the  influence  of  neutral  bodies,  inorganic  salts  or  proteids, 
in  diminishing  disinfectant  action  by  decreasing  dissociation. 

The  problem  is,  of  course,  complicated  by  still  other  chemical 
interactions  which  are  more  obscure.  For  example,  Scheurlen,  (1895), 
Beckmann  (1896),  Romer  (1898),  and  Spiro  and  Bruns  (1898)  have 
shown  that  in  the  case  of  phenol  and  certain  other  organic  disinfectants 
the  addition  of  sodium  chloride  greatly  increases  toxic  action.  Si  ill 
another  factor  which  affects  disinfectant  power  has  been  brought 
out  in  recent  years  by  Nageli  (1893),  and  other  observers — the 
presence  of  suspended  solid  particles  of  neutral  character.  In 
the  most  recent  communication  upon  this  subject  by  True  and 
Oglevee  (1905)  it  was  shown  that  the  toxic  effect  of  metallic  salts 
upon  Lupiniis  may  be  entirely  counteracted  by  the  presence  of 
finely  divided  particles  of  sand,  glass,  filter  paper,  coal,  starch,  or 
paraffm.     On  the  other  hand, the  toxic  effect  of  organic  disinfectants 


266  C.-E.  A.  WiNSLOW  AND   E.  E.  LOCHRIDGE 

— phenol,  thymol,  and  resorcinol — was  affected  only  to  a  barely 
appreciable  degree.  The  action  when  it  occurs  is  explained  by  the 
power  of  suspended  particles  to  remove  dissolved  substances  by  a 
process  of  adsorption,  and  the  possibility  suggests  itself  that  the 
removal  of  ions  by  large  organic  molecules  in  true  or  colloidal 
solutions  may  be  of  an  analogous  character.  Whatever  the  cause, 
this  phenomenon  must  prove  of  far-reaching  importance  in  bacteri- 
ology. Such  facts  as  the  observed  multiplication  of  bacteria,  when 
water  samples  are  stored  in  glass  bottles,  may  be  the  result  of  a 
removal  of  inhibiting  substances  by  the  adsorptive  action  of  glass 
surfaces. 

A  considerable  body  of  evidence  in  the  field  of  anim.al  physiology 
bears  out  these  conclusions  obtained  from  the  study  of  bacteria  and 
other  plants.  A  fairly  full  summary  of  this  literature  may  be  found 
in  the  reviews  of  Cohen  (1903)  and  Hamburger  (1904).  The  work 
of  Kahlenberg  (1898)  and  other  observers,  who  have  shown  that  the 
taste  of  dilute  solutions  is  in  many  cases  due  to  the  specific  properties 
of  the  dissociated  ions,  is  of  interest.  Studies  which  have  been 
frequently  cited  were  made  by  Loeb  (1897  and  1898),  and  recently 
reprinted  (Loeb,  1905),  on  the  influence  of  free  ions  upon  frog's 
muscle.  The  gastrocnemius  muscle  absorbs  water  and  increases  its 
weight  in  the  presence  of  slight  traces  of  acids  or  alkalis,  and  Loeb  con- 
cluded that  for  the  inorganic  acids  and  bases  this  increase  in  weight 
is  solely  a  function  of  the  number  of  hydrogen  and  hydroxyl  ions  in 
the  unit  of  volume.  This  sweeping  conclusion  is  hardly  borne  out 
by  his  experiments.  With  the  organic  acids  there  was  no  relation 
whatever.  Trichloracetic  acid,  almost  entirely  dissociated,  and 
lactic  acid,  with  only  11  per  cent  dissociation,  gave  practically  the 
same  results.  With  a  series  of  11  different  organic  acids  of  every 
degree  of  dissociation,  the  individual  variation  in  weight-increase, 
with  0.009  normal  solutions,  ranged  only  between  3.9  per  cent  and 
7.2  per  cent. 

A  very  significant  line  of  physiological  investigation  concerns  the 
binding  of  free  ions  by  organic  molecules  of  large  size.  In  one  of 
the  most  recent  communications  on  this  subject,  Stiles  and  Beers 
(1905)  have  shown  that  the  effect  of  calcium  chloride,  barium  potas- 
sium chloride,  and  sodium  nitrite  upon  plain,  cardiac,  and  striped 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       267 

muscle  of  the  frog,  terrapin,  and  guinea-pig  was  reduced  from  one 
half  to  three-fourths  in  the  presence  of  white  of  egg,  partially  dialyzed 
serum,  peptone,  or  starch.  In  some  cases  a  combination  between  the 
inorganic  body  and  the  proteid  has  been  demonstrated  by  freezing- 
point  determinations,  but  in  other  cases  particularly  with  the  neutral 
salts,  this  has  not  been  shown.  As  the  authors  suggest,  these  experi- 
ments point  to  the  existence  of  ^^physiological  compounds  which  are 
not  demonstrable  at  all  by  chemical  methods,  but  only  by  the 
reactions  of  living  tissues." 

2.     OBJPXT  AND  METHODS  OF  THE  PRESENT  INVESTIGATION. 

The  present  investigation  was  begun  with  the  intention  of  deter- 
mining the  effect  of  acid  wastes  in  sewage  upon  the  viability  of  the 
typhoid  bacillus  under  practical  conditions.  It  soon  appeared, 
however,  that  the  problem  was  too  complex  to  be  attacked  in  any 
general  way  without  the  preliminary  determination  of  certain  of  the 
individual  factors  involved,  under  definitely  controlled  conditions. 
We  have  therefore  attempted  to  find  the  disinfectant  power  of  tw(~ 
mineral  acids  and  two  organic  acids  upon  the  typhoid  bacillus  in  tap 
water  and  in  the  presence  of  peptone,  and  have  controlled  these 
experiments  by  a  parallel  series  wdth  the  colon  bacillus.  The  results, 
besides  their  specific  value  as  determinations  of  the  reactions  of  these 
two  organisms  to  dilute  acids,  have  a  certain  interest  in  relation  to 
the  general  theory  of  disinfection.  In  all  the  experiments  reviewed 
above,  except  Dial's,  the  acids  used  were  tested  in  only  a  few  widely 
differing  strengths,  so  that  the  parallelism  between  disinfectant  action 
and  dissociation  was  not  established  with  any  great  exactness.  In 
the  work  of  Kronig  and  Paul  on  anthrax  spores  and  the  various  studies 
on  the  mold  fungi,  it  was  necessary  to  use  such  strong  solutions 
that  ionic  effects  were  largely  masked  by  the  influence  of  the  undis- 
sociated  molecule,  and  in  the  studies  of  Kahlenberg  and  True  and 
Heald  on  the  phanerogams  it  was  evident  that  with  such  complex 
organisms  many  other  factors  than  the  direct  effect  on  protoi)lasm 
come  into  play.  There  was  room,  therefore,  for  a  series  of  experi- 
ments on  organisms  sensitive  to  very  dilute  acids,  carried  out  in 
suffK-ient  detail  to  show  definitely  the  relations  between  toxicity 
and  dissociation. 


268  C.-E.  A.  WiNSLOW   AND   E.  E.  LOCHEIDGE 

With  the  view  of  securing  exact  quantitative  resuhs,  we  adopted 
the  method  of  exposure  to  the  acid  tested,  in  a  suspension  from 
which  samples  were  directly  plated.  This  process  has  the  obvious 
defect  of  permitting  a  certain  amount  of  the  disinfectant  to  be 
carried  over  to  the  plate,  where  it  may  exert  an  antiseptic  action. 
We  have  really  measured  the  combined  disinfectant  action  in  the 
suspension  and  possible  antiseptic  action  in  the  plate.  The  action 
of  organic  matter  in  decreasing  toxicity,  shown  by  our  experiments, 
must  greatly  reduce  any  such  action  in  the  plate. 

The  procedure  in  each  experiment  was  as  follows:  A  series  of 
bottles,  each  containing  loo  c.c.  of  sterile  water  (or  peptone  solution), 
was  arranged  in  a  row,  and  to  each  bottle  was  added  a  different 
amount  of  standardized  acid  from  a  graduated  pipette.  The  amount 
of  water  in  each  bottle  was  measured  at  the  end  of  the  experiment, 
in  order  to  obtain  the  exact  strength  of  the  solution.  Immediately 
after  the  addition  of  the  acid  there  was  added  to  each  bottle  i  c.c.  of 
a  fresh  aqueous  suspension  of  the  bacteria  tested.  After  standing 
for  40  minutes,  lactose  agar  plates  were  made  in  duplicate,  from 
the  acidified  bottles,  and  from  controls  with  no  acid.  Colonies  were 
counted  after  24  hours'  incubation  at  37°  C. 

Forty  minutes  was  selected,  after  some  preliminary  experiments, 
as  the  best  period  of  exposure  to  the  acid,  since  it  gave  sharper 
results  than  a  shorter  time.  In  the  tests  reported  in  the  accompany- 
ing tables  there  was  not  a  variation  from  the  40  minutes  of  more  than 
one  minute  in  most  of  the  samples.  The  series  for  B.  typhi  in  water, 
with  HCl,  were  also  examined  after  100  minutes,  and  after  24  hours. 
In  the  sample  containing  48  parts  per  million  of  sulphuric  acid, 
there  was,  after  40  minutes,  59.3  per  cent  removal  of  B.  typhi;  after 
100  minutes,  88.15  per  cent  removal;  after  24  hours,  100  per  cent 
removal.  The  sample  containing  92.9  parts  per  million  of  sulphuric 
acid  removed  after  40  minutes  92.97  per  cent;  after  100  minutes, 
99.99+  per  cent ;  after  24  hours,  100  per  cent.  The  removal  was  100 
per  cent  in  all  of  the  samples  containing  larger  amounts  of  acid  after 
103  minutes,  and  in  all  of  the  samples  after  24  hours. 

The  temperature  factor  was  of  considerable  importance,  and  care 
was  taken  to  keep  conditions  uniform.  No  agar  was  poured  with  a 
temperature  greater  than  50°  C.     It  was  found  that  a  rise  in  tem- 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       269 

peralure  in  the  presence  of  the  acid  was  very  fatal  in  its  efTect  on  the 
bacteria. 

The  typhoid  cuUure  used  was  obtained  from  the  Massachusetts 
General  Hospital,  where  it  had  been  isolated  from  the  spleen  of  a 
clinically  typical  case  of  typhoid  fever;  the  colon  bacillus  was  isolated 
in  the  laboratories  of  the  Institute,  and  both  gave  all  characteristic 
reactions.  Twenty-four-hour  agar-slant  cultures  were  used  in  all 
cases. 

The  tables  have  been  prepared  to  show,  in  the  first  column,  the 
parts  per  million  of  the  acid,  and  in  the  second  column  the  parts  per 
million  of  acidic  hydrogen  or,  more  accurately,  of  rej)laceable 
hydrogen.  The  third  column  shows  the  strength  converted  into  terms 
of  normality.  The  percentage  dissociation  of  the  acids  at  each  dilu 
tion  is  given  in  the  fourth  column,  and  the  actual  parts  of  disso- 
ciated hydrogen  in  the  fifth.  The  last  two  columns  show  the  initial 
number  of  bacteria  used  as  shown  by  blank  controls  and  the  per- 
centage reduction  after  40  minutes. 

The  tables  show  in  general  that  with  increasing  quantities  of 
disinfectant  the  bacterial  reduction  proceeds  rapidly  up  to  a  certain 
point.  After  99  per  cent  of  the  organisms  have  been  killed,  how- 
ever, it  takes  a  very  considerable  further  increase  of  acid  to  produce 
sterilization.  This  is  a  point  of  ver\'  fundamental  importance,  and 
one  which  has  been  observed  in  studying  the  elTect  of  such  various 
agents  upon  the  bacteria,  that  it  deserves  special  attention.  Sedg- 
wick and  one  of  us  (Sedgwick  and  Winslow,  1902)  have  called  atten- 
tion to  the  persistence  of  a  few  specially  resistant  individuals  when 
typhoid  bacilli  are  exposed  to  the  action  of  cold.  After  14  days  of 
exposure  to  freezing  temperature  99.8  per  cent  of  the  organisms 
were  killed,  but  after  three  months  a  few  still  survived.  Johnson's 
tables  of  the  reduction  of  typhoid  and  colon  bacilli  by  copper  salts 
(Johnson,  1905)  show  the  same  phenomenon,  although  he  d(KS 
not  comment  upon  it  specifically.  More  recently.  Frost  and  Swenson 
(1906),  and  Gage  and  Stoughton  (1906),  have  cmj)hasized  this 
peculiar  phenomenon,  in  connection  with  resistance  to  high  tem- 
peratures. The  former  authors,  working  with  B.  dyscnteriae,  found 
that  "the  majority  of  the  cells  were  killed  between  55°  and  60°,  but 
that  frequently  a  relatively  small  number,  possibly  one  individual 


270  C.-E.  A.  WiNSLOW  AND   E.  E.  LOCHRIDGE 

in  a  hundred  thousand  or  a  million,  may  persist  at  much  higher 
temperatures,  even  70°."  Gage  and  Stoughton  in  their  conclusions 
point  out  that  "the  great  majority  of  the  bacteria  in  any  B.  coli 
cultures  are  destroyed  by  five  minutes'  exposure  to  some  tempera- 
ture between  50°  and  60°  C.  A  few  individuals,  however,  in  each 
culture  will  survive  much  higher  temperatures,  in  some  cases  re- 
maining alive  after  exposure  to  90°  C.  The  very  close  range  (about 
10°  C.)  of  temperature  at  which  the  destruction  of  the  majority  of  the 
individual  bacteria  occurred,  as  compared  with  the  considerable 
range  (about  35°  C),  in  the  temperatures  at  which  complete  sterili- 
zation was  effected  would  indicate  that  the  determination  of  the 
majority  death-point  would  be  of  more  value  in  species  identification 
than  is  the  determination  of  the  absolute  thermal  death-point  as  at 
present  employed." 

Altogether  it  seems  clear  that  among  what  are  ordinarily  con- 
sidered non-sporing  bacteria  there  exists  a  small  proportion  of  indi- 
viduals having  specially  high  resistant  powers  against  unfavorable 
conditions.  The  absolute  death-point  for  these  resistant  forms  is 
difficult  to  determine  accurately  on  account  of  their  small  numbers 
and  the  consequent  chances  that  they  may  be  overlooked.  We  are 
inclined  from  our  experience  to  agree  with  Gage  and  Stoughton  as 
to  the  superior  value  for  many  purposes  of  the  majority  death-point 
(99  per  cent),  and  we  shall  lay  special  stress  on  this  in  interpreting 
our  results. 

3.     THE  DISINFECTANT  ACTION  OF  HYDROCHLORIC  ACID  AND 
SULPHURIC  ACID  UPON  B.   TYPHI  AND  B.  COLI. 

Hydrochloric  acid  and  sulphuric  acid  were  chosen  as  types  for 
the  study  of  strong  mineral  acids,  and  the  experiments  were  carried 
out  as  described  above.  The  water  used  was  Boston  tap  water, 
containing  before  sterihzation  about  40  parts  per  million  of  residue, 
15  parts  of  hardness,  0.015  P^i"t  ^^  free  ammonia,  and  0.144  parl  of 
albuminoid  ammonia.     The  results  are  shown  in  Tables  1-4. 

The  99  per  cent  killing-point  with  the  hydrochloric  acid  is  reached 
at  a  strength  of  0.0077  normal,  with  7.49  parts  of  dissociated  hydro- 
gen per  million,  and  the  absolute  killing-point,  as  nearly  as  it  can  be 
determined,  with  a  0.0123  normal  solution  containing  11.80  parts 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli 


271 


TABLE  1. 
Action  of  Hydrochloric  Acid  on  B.  colt  in  Tap  Water. 


Acid  Parts  in 
1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  l)e(orc 
Treatment 

Percentage  Re- 
duction of  Barlrria 
after  40  Min. 

38.1 
77-6 
104  5 
140.0 
178.9 
281.0 
298.0 
377  0 
447.0 
515  0 
590.0 
663.0 

I 
2 
3 

4 
4 
7 
8 
10 
12 

05 
13 

^2 
08 

90 

71 

17 

61 

26 

0.0010 
0 .0021 
0.0033 
0  0041 
0 .0049 
0  0077 
0.0082 
0  0106 
0.0123 

98 
98 
97 
97 
97 
97 
96 
96 
96 
96 
96 
96 

0 
0 
5 
3 
2 
0 
5 
4 
4 
3 
3 

I  03 
2.00 
3    24 
3.98 
4.76 
7   49 
7.90 
10.  24 
1 1    80 

40,000 

00,000 
It 

00  00 

45  00 

72  8s 

82.50 

07  50 

00  87 

00  06 

00  00 

100  00 

100.00 

100.00 

100   00 

TABLE  2. 
Action  of  Sulphuric  Acid  on  B.  colt  in  Tap  Water. 


Acid  Parts  in 
1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 

c.c.  before 

Treatment. 

Percentage  Re 

duction  of  Bacteria 

after  40  Min. 

45.9 

0.94 

0  0009 

93 

0.87 

60,000 

66.20 

46.5 

0.95 

0.0009 

93 

0.87 

140,000 

80.00 

111.5 

2.28 

0.0023 

90 

2.05 

*' 

81.43 

138.3 

2.82 

0.0028 

89 

251 

60,000 

86  so 

176.6 

3.62 

0.0036 

88 

3.18 

" 

96.25 

178.2 

3.64 

0.0036 

88 

3  20 

140,000 

82.14 

225.2 

4.59 

0 . 0046 

86 

3-94 

60.000 

06.2s 

2572 

5.25 

0.0052 

?5 

4.46 

" 

98.  so 

311-5 

6.36 

0.0064 

84 

5-35 

140,000 

07  50 

375-9 

7.68 

0  0077 

83 

6.38 

q8  86 

470.0 

9.60 

0 . 0096 

80 

7.68 

'* 

00  92 

552  0 

11.30 

0.0113 

79 

8.93 

* 

00  02 

630.0 

12.90 

0 .0129 

78 

10.05 

" 

09  95 

692.5 

14.13 

0.0141 

77 

10  .90 

" 

90  OS 

812.0 

16.57 

0.0166 

76 

12.60 

'* 

100  00 

910.0 

18. 57 

0.0186 

75 

13   95 

100  00 

of  dissociated  hydrogen.  With  the  sulphuric  acid  the  99  per  cent 
reduction  was  reached  at  a  strength  of  0.0096  normal,  with  7.68  jiarts 
of  dissociated  hydrogen,  and  the  100  per  cent  reduction  at  a  strength 
of  0.0166  normal,  with  12.6  parts  of  dissociated  hydrogen.  These 
results  show  a  direct  relation  between  disinfectant  action  and  free 
hydrogen  ions.  The  normal  strengths  of  the  killing  solutions  do  not 
correspond  very  closely;  0.0077  sulphuric  acid  failed  to  do  what  the 
same  strength  of  hydrochloric  acid  did,  and  0.0129  and  0.0141  sul- 
phuric acid  failed  to  do  what  0.0123  hydrochloric  acid  did.  On  the 
other  hand,  when  we  compare  dissociated  hydrogen,  allowing  for 
the  greater  ionization  of  the  hydrochloric  acid,  the  discrepancies 
disappear.     The  same  concentrations  of  dissociated  hydrogen,  within 


272 


C.-E.  A.  WiNSLOW    AND    E.  E.  LOCHRIDGE 


the  limits  of  accuracy  of  the  experiment,  produced  respectively  the 
99  per  cent  and  the  100  per  cent  reduction  in  the  two  acids. 

TABLE  3. 
Action  of  Hydrochloric  Acid  on  B.  typhi  in  Tap  Water. 


Acid  Parts  in 
1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Percentage  Re- 
duction of  Bacteria 
after  40  Min. 

38.2 

1 .  00 

O.OOIO 

98.2 

1 .02 

7,050 

79.20 

75. 3 

2  .06 

0 . 002  T 

97 

9 

2  02 

88.10 

109.5 
I48.2 

3.00 
4.06 

0 . 0030 
0.0041 

97 
97 

8 
3 

2  94 

3  95 

99.30 
99.86 

182.  s 

5.00 

0 . 0050 

97 

I 

4   85 

100.00 

216.8 
248.0 

5  92 
6.80 

0.0059 
0 . 0068 

100.00 
100.00 

275.5 

7-54 

0.0075 

100.00 

TABLE  4. 
Action  of  Sulphuric  Acid  on  B.  typhi  in  Tap  Water. 


Acid  Parts  in 
1 ,000,000 

Hydrogen 
Parts  in 
I  000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Percentage  Re- 
duction of  Bacteria 
after  40  Min. 

48.0 

92.9 

139  4 

188.9 

223.0 

0,98 
1.88 
2.84 
385 

4-55 

O.OOIO 

0.0019 
0 . 0028 
0.0038 
0 . 0045 

93-1 
90.5 
89.5 
87.0 

85.5 

0.91 
r.70 
2-54 
3-35 
3   90 

135,000 

59.30 
92.97 
99.99 
96. 10 
100.00 

Tables  3  and  4  show  that  the  typhoid  bacillus  is  considerably 
more  sensitive  than  the  colon  bacillus  in  its  reaction  to  an  excess  of 
acid.  The  99  per  cent  reduction  was  reached  with  hydrochloric 
acid  at  a  strength  of  0.0030  normal  and  2.94  parts  of  dissociated 
hydrogen,  and  the  100  per  cent  reduction  with  0.0050  normality  and 
4.85  parts  of  dissociated  hydrogen.  With  sulphuric  acid  the  99  per 
cent  reduction  was  reached  with  0.0028  normality  and  2.54  parts  of 
hydrogen,  and  the  100  per  cent  reduction  with  0.0045  normality 
and  3.90  parts  of  hydrogen.  The  fact  that  the  0.0038  normal  solution 
of  sulphuric  acid  showed  only  96  per  cent  reduction  is  one  of  the 
abnormalities  which  unfortunately  sometimes  occur  in  bacteriological 
work.  In  general,  the  results  show  again  that  the  two  acids  exert 
the  same  quantitative  effect,  although  in  this  case,  the  solution  being 
weaker  and  the  dissociation  of  the  two  acids  more  nearly  the  same, 
the  difference  between  normal  strength  and  concentration  of  dis- 
sociated hydrogen  is  not  clearly  shown. 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli      27^ 

The  critical  points  derived  in  these  tests  are  brou-^lil  loj^ether  in 
Table  5.  They  show  that  the  typhoid  badllus  is  a  little  less  than  half 
as  resistant  as  the  colon  bacillus  to  dilute  acids,  and  that  the  toxicity 
of  these  acids  depends,  not  on  their  normal  strength  of  acid  or  on  the 
kind  of  acid  used,  but  on  the  number  of  dissociated  hydrogen  ions. 
Between  7.4  and  7.7  parts  of  dissociated  hydrogen  effects  a  gg  per  cent 
reduction  of  the  colon  bacillus,  and  between  11. 8  and  12.6  parts,  a 
100  per  cent  reduction.  For  the  typhoid  bacillus  the  corresponding 
figures  are  2.5-3.0  parts  and  3.g-4.g  parts.  Since  at  the  dilutions 
used  the  hydrochloric  acid  was  over  g6  per  cent  dissociated,  its 
effect  must  have  been  almost  entirely  ionic;  and  since  the  sul- 
phuric acid  at  75  per  cent  dissociation  showed  only  the  to.xicity  which 
would  have  been  expected  from  its  dissociated  hydrogen,  it  appears 
that  in  this  case  too  the  undissociated  molecule  exerts  no  appreciable 
influence.  The  anions  have  been  shown  to  be  neutral  in  the  experi- 
ments of  other  observers.  It  is  evident,  then,  that  the  toxicity  of  those 
acids  at  high  dilution  is  a  function  of  the  dissociated  hydrogen. 

TABLE  5. 
Disinfectant  Action  of  Mineral  Acids  in  Tap  Water. 


B.  coli 

B.  typhi 

09%  Reduction 

100%  Reduction 

09%  Reduction 

100%  Reduction 

HCl 

H,SO« 

HCl 

H.SO4 

HO 

H.SO4 

HCl 

H.SO, 

Normality 

Parts  per    1,000,000   dis- 
sociated hydrogen 

0.0077 
7.40 

o.oog6 
7.68 

0.0123 
12.80 

0.0166 
12.60 

0.0030 
2.94 

0.0028 
a   S4 

0  0050 
4.8s 

0  004S 
3  00 

4.     THE  DISINFECTANT  ACTION  OF  ACETIC  ACID  AND   BENZOIC 
ACID  UPON  B.    TYPHI    AND  B.  COLI. 

We  next  desired  to  study  examples  of  the  incompletely  dissociated 
organic  acids.  Acetic  and  benzoic  acids  were  selected  .as  types, 
and  the  experiments  were  carried  out  as  before.  The  results  ob- 
tained with  benzoic  acid  are  probably  somewhat  inaccurate  on 
account  of  the  difficulty  of  securing  complete  solution.  The  results 
are  shown  in  Tables  6-8. 

An  inspection  of  these  tables  shows  a  marked  difference  from  the 
results  obtained  with  the  mineral  acids.  With  B.  coli  in  acetic  acid  the 
gg  per  cent  reduction  is  reached  at  a  strength  of  0.0812  normal,  and 


274 


C.-E.  A.  WiNSLOW   AND    E.  E.  LOCHRIDGE 


TABLE  6. 
Action  of  Acetic  Acid  on  B.  coli  in  Tap  Water. 


Acid  Parts 
in  1,000,000 

Hydrogen 
Parts  in 

1,000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Percentage    Re- 
duction of  Bac- 
teria after  40 
Min. 

III. 3 

1.85 

0.0018 

.... 

60,000 

00.00 

333 -o 

5-54 

0.0055 

6.40 

0.35 

" 

00.00 

552.0 

9.18 

0.0092 

4.50 

0 .41 

" 

16.67 

732.0 
1,095.0 
1,386.0 

12  .20 
18.25 
23.20 

0.0122 
0.0182 
0.0232 

3   50 
3  05 
2.75 

0.43 
0.  56 
0.64 

" 

23 -33 
38.33 
51-67 

1,825.0 
2,260.0 
3,081  0 
3,698  0 
4,800.0 

30.41 
37.70 
51.40 
61.  70 
80.20 

0.0304 
0.0377 
0.0514 
0.0617 
0.0802 

2.45 
2.  20 
1.90 
1.70 
I    50 

0.75 
0.83 
0.98 
105 
1.20 

ti 

SS-oo 
56-67 
58.33 
63 -33 
91 .00 

4-875.0 

81.25 

0.0812 

I    50 

1 .  21 

90,000 

99  99 

5,610.0 

93   50 

0.0935 

1-35 

1.26 

100.00 

TABLE  7. 
Action  of  Benzoic  Acid  on  B.  coli  in  Tap  Water. 


Acid  Parts 
in  1,000,000 

Hydrogen 
Parts  in 

1,300,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Percentage   Re- 
duction of  Bac- 
teria after  40 
Min. 

237.9 
3370 
406.0 
675-0 
1,184.0 
2,425  0 

1-95 
2-76 
3-33 
5-54 
9-72 
19.90 

0-0019 
0 . 0028 
0.0033 
0.0055 
0.0097 
0. 0199 

16.0 
13-6 

12-5 

9   3 

7-5 
5-4 

0.31 
0.38 
0.41 
0.52 
0.73 
1.07 

70,000 

" 
100,000 

00.00 
50.00 
60.75 
67.80 
99  99 
100.00 

TABLE  8. 
Action  of  Benzoic  Acid  on  B.  typhi  in  Tap  Water. 


_  Add  Parts 
in  1,000,000 


Hydrogen 
Parts  in 
1,000,000 


Normality 


Per  Cent  of 
Dissociation 


Dissociated 

Hydrogen. 

Parts  in 

1,000,000 


Bacteria  per 
c.c.  before 
Treatment 


Percentage   Re- 
duction of  Bac- 
teria after  40 
Min. 


29.23 
140.30 
243 . 80 
333.00 
42  7 . 00 
689 . 00 
1,292.00 


.24 
15 

.00 
73 

■52 
75 
10.60 


0.0002 
0.0011 
0.0020 
o . 002  7 
0.0035 
0.0057 
0.0106 


40.0 
20.3 
15-4 
13-6 
10.4 
9.8 

7-2 


10 

23 
31 
37 
37 
56 
76 


50,000 


44.00 
54.00 
62.00 
70.00 
92  .00 
100.00 
100.00 


the  100  per  cent  reduction  at  0.0935  normal.  The  acid  at  these 
strengths  is  only  a  little  over  i  per  cent  dissociated,  and  the  amount 
of  dissociated  hydrogen  present  a  little  over  1.2  parts  per  million. 
Since  this  is  only  about  one-sixth  the  strength  of  ionic  hydrogen 
necessary  to  produce  similar  results  with  the  mineral  acids,  it  is  evi- 
dent that  the  toxic  action  of  the  acetic  acids  is  due  chiefly  to  the  anion 
or  the  undissociated  molecule,  the  latter,  being  so  much  greater  in 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       ^75 


amount,  probably  playing  the  principal  part.  The  same  thing  is 
true  of  benzoic  acid.  Here  the  molecule  is  more  highly  toxic, 
producing  a  99  per  cent  reduction  at  0.0097  normality  and  a  100  per 
cent  reduction  at  0.0199  normality,  with  about  i  per  cent  dissocia- 
tion. As  in  the  case  of  the  mineral  acids  B.  typhi  is  more  sensitive 
than  B.  coli,  showing  100  per  cent  reduction  with  benzoic  acid  at  a 
strength  of  0.0057  normal. 

It  appears,  then,  that  the  toxicity  of  these  organic  acids  is  due  not 
mainly  to  hydrogen  ions,  but  to  the  action  of  the  undissociated 
molecule,  varying  widely,  as  might  be  expected,  with  the  acid 
employed.       A    comparison    of    the    corresponding    toxic    normal 

TABLE  9. 

Toxicity   of   Organic   and   Mineral   Acids   for    B.  coli  and  B.  typhi.  Strength    in  Normality 

Producing  99  Per  Cent  and  100  Per  Cent  Reduction. 


B.  coli 

B.  typhi 

Acid 

99%  Reduction 

ioo%  Reduction 

99%  Reduction 

100%  Reduction 

Hydrochloric 

Sulphuric 

0.0077 
0.0096 
0.0812 
0.0097 

0.0123 
0.0166 
0.0935 
0. 0199 

0.0030 
0.0028 

0.0050 
0.0045 

Acetic 

Benzoic 

O.OOS7 

strength,  made  in  Table  9,  shows  that  benzoic  acid  is  almost  as 
toxic  as  the  mineral  acids,  the  effect  being  due  in  one  case  to  the 
whole  molecule,  and  in  the  other  case  to  hydrogen  ions.  Acetic 
acid,  on  the  other  hand,  has  only  10-20  per  cent  as  high  a  disinfect- 
ant action. 


5.    THE  DIMINISHED  TOXICITY  OF  ACIDS  IM  THE    PRESENCE 

OF  PEPTONE. 

Having  fixed  with  some  precision  the  killing-point  for  the  various 
acids  studied,  when  acting  in  tap  water,  we  next  desired  to  determine 
what  would  occur  in  the  presence  of  organic  matter.  .\  series  of 
experiments  was  carried  out,  parallel  to  those  reported  above,  except 
that  a  I  per  cent  solution  of  Witte's  peptone  was  used  instead  of  tap 
water.  The  results  with  the  mineral  acids,  presented  in  Tables  10-12, 
showed  that  the  toxicity  of  the  acid  is  profoundly  modified  by  the 
presence  of  organic  matter. 


276 


C.-E.  A.  WiNSLOW   AND    E.  E.  LOCHRIDGE 


TABLE   10. 
Disinfectant  Action  of  Hydrochloric  Acid  on  B.  coli  in  i  per  Cent  Peptone  Solution. 


Acid  Parts  in 

1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent 

of 

Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Perceritage 

Reduction  of 

Bacteria  after 

40  Min. 

1,1x8 

30.62 

0.0306 

94.8 

29.0 

90,000 

40.00 

1,82s 

50.00 

0.0500 

94  8 

47.  5 

" 

93   35 

2,502 

69.00 

0.0690 

93   0 

64.2 

'* 

97  97 

2,950 

80.80 

0 . 0808 

92  0 

74-4 

" 

97-74 

3.470 

95   20 

0.0952 

91-5 

87.5 

" 

99  98 

3.685 

97.80 

0.0978 

91    4 

89.4 

'* 

100  00 

4,020 

110.00 

0.  IIOO 

100 . 00 

TABLE   ir. 
Disinfectant  Action  of  Sulphuric  Acid  on  B.  coli  in  i  per  Cent  Peptone  Solution. 


Acid  Parts 
in 

1,000,000 

Hydrogen 
Parts  in 
1 ,000,000 

Normality 

Per  Cent 

of 

Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

Bacteria  per 
c.  c.  before 
Treatment 

Percentage 

Reduction   of 

Bacteria  after 

40  Min. 

1,000,000 

970 

140,000 

00 .00 

1,204 

" 

00.00 

1.40S 

00.00 

1-536 

314 

0.0314 

70 

0 

22 

0 

" 

00.00 

1,728 

35-3 

0.0353 

68 

5 

24 

2 

" 

20 .00 

1,962 

40. 1 

0.0401 

67 

5 

27 

I 

'* 

61.43 

2,066 

40 -9 

0.0409 

67 

0 

27 

4 

'* 

64.29 

2,399 

48.9 

0.0489 

6s 

4 

32 

0 

60,000 

76.67 

2,639 

53-8 

0  0538 

64 

8 

24 

9 

" 

79-17 

2,912 

59.4 

0.0594 

64 

2 

38 

I 

" 

91-67 

3.065 

62.6 

0.0626 

63 

2 

30 

6 

'* 

93   33 

3.258 

66.5 

0 . 0665 

62 

8 

41 

8 

'* 

94   17 

3.450 

70.4 

0 .0700 

61 

9 

43 

5 

" 

98.85 

4.610 

94   2 

0.0942 

58 

9 

55 

5 

65,000 

99   99 

5.298 

108. 1 

0. 108 I 

57 

0 

61 

6 

'* 

100.00 

6,555 

135-2 

0.1352 

'* 

100 .00 

7,800 

159-3 

0  -  1593 

loo. 00 

TABLE  12. 
Disinfectant  Action  of  Hydrochloric  Acid  on  B.  lyphi  in  i  per  Cent  Peptone  Solution. 


Acid  Parts 
in 

1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissociation 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria  per 
c.c.  before 
Treatment 

Percentage 

Reduction  of 

Bacteria  after  40 

Min. 

1,107 
1,740 

30.3 
47  6 

0.0330 
0.0476 

94-8 
93-8 

28.7 
44-6 

50,000 
50,000 

99-99 

100.00 

In  these  tables  the  dissociation  values  given  are  those  determined 
for  distilled  water,  and  not  those  v^hich  actually  obtain  in  a  peptone 
solution.  The  amount  of  dissociated  hydrogen  required  for  disin- 
fection, when  estimated  in  this  way,  is  seen  to  be  nearly  lo  times  as 
great  as  in  tap  water.  For  comparison  the  results  are  brought 
together  in  Table  14. 


Effect  of  Acids  on  Typhoid  and  Colon  B.xcilij 


TABLE  13, 
Disinfectant  Action  of  Sulphuric  Acid  on  b.  typhi  in  i  per  Cent  Peptone  Solution. 


Acid  Parts 

in 
1,000,000 

Hydrogen 
Parts  in 
1 ,000,000 

Normality 

Per  Cent 

of 

Dissociation 

Dissociatrd 

HydroRci.. 

Parts  in 

1,000,000 

Bacteria  pc-r 
c.  c.  before 
Treatment 

Pcrftntagr 

Reduction   of 

Bacteria  after 

40  Min. 

819 

17.03 

0.0170 

74-5 

12.7 

85,000 

00.00 

960 

20.00 

0.0200 

73-7 

14   7 

1 7  6s 

1,104 

22.52 

0.0225 

72.0 

16.2 

'* 

17  65 

1. 1^0 

23.17 

0.0232 

72.0 

16.7 

" 

34   'O 

1,288 

26.32 

0.0265 

71.0 

18.7 

*' 

61 .20 

l>453 

20  63 

0.0296 

70.2 

20.8 

28,000 

82.15 

1,472 

30.03 

0.0300 

70.1 

ai.o 

85,000 
28,000 

76.4s 

1,510 

31-66 

0.0317 

69.7 

21.6 

80  30 

■.587 

32.39 

0.0324 

69.5 

22.5 

'* 

80  30 

T,6l2 

32  89 

0.0329 

69.0 

22.7 

" 

100  00 

1,678 

34   24 

0.0342 

68.5 

23  4 

" 

07  86 

1.879 

38.32 

0.0383 

68.0 

26.0 

'* 

100  00 

1,994 

'* 

loo  00 

2,045 

*' 

100  00 

2,115 

100  00 

2,119 

•  1 

100  00 

2,399 

100  00 

TABLE  14. 
Comparative  Toxicity  of  Mineral  Acids  in  Distilled  Water  and  i  per  Cent  Peptone  Souttion. 

(Parts  per  1,000,000  of  dissociated  hydrogen.) 


B.  colt 

B.  typhi 

99%  Reduction 

100%  Reduction 

99%  Reduction 

100%  Reduction 

HCl 

H,S04 

HCl 

H,S04 

HCl 

H,S04 

HCl 

H,SO« 

Distilled  water 

7.49 
87. 5 

7.68 
5SS 

11.80 
89.4 

12.60 
61.6 

2.94 
28.7 

2-54 

4.85 
44  6 

3   90 

I  per  cent  peptone 

22.  7 

It  is  evident  that  in  some  way  the  peptone  exerts  a  strong  inlluencc 
in  counteracting  the  toxic  effect  of  the  acids.  It  at  first  occurred  to 
us  that  this  might  be  due  simply  to  the  fact  that  peptone  solution 
furni.shed  a  more  favorable  medium  for  the  bacteria,  and  thus  enabled 
them  to  resist  unfavorable  conditions.  Such  an  effect  would,  how- 
ever, hardly  be  expected  in  so  short  a  period  as  40  minutes;  and  this 
explanation  fails  to  account  for  the  fact  that  the  toxicity  of  the  hydro- 
chloric acid  is  much  more  diminished  than  that  of  the  sulphuric  acid. 
Reference  to  Tables  15-17,  which  show  the  results  obtained  with  the 
organic  acids,  makes  it  still  clearer  that  a  specific  chemical  action  is 
involved. 

Evidently  with  the  organic  acids  disinfectant  power  is  much 
less  affected  by  the  presence  of  peptone.  With  B.  coli  acetic  acid 
produces  a   100  per  cent  reduction  when   in  a  strength  of  0.0935 


278 


C.-E.  A.  WiNSLOW   AND   E.  E.  LOCHRIDGE 


TABLE  IS. 
Disinfectant  Action  of  Acetic  Acid  on  B.  colt  in  i  per  Cent  Peptone  Solution. 


Acid  Parts 
in  1,000,000 

Hydrogen 
Parts  in 

Normality 

Per  Cent  of 
Dissocia- 

Dissociated 

Hydrogen. 

Parts  in 

Bacteria 

per  c.c. 

before 

Percentage    Re- 
duction of  Bac- 
teria after  40 

1,000,000 

1,000,000 

Treatment 

Min. 

4.350 

72.  S 

0.0725 

1.60 

1. 16 

50,000 

00.00 

4.7SO 

79-3 

0.0793 

1.50 

1. 19 

00.00 

S.340 

8q.o 

0 . 0890 

1.40 

I  25 

25.00 

5.97S 

97.0 

0.0970 

1.30 

1.26 

66.00 

5.995 

98.0 

0.0980 

1-30 

1.27 

61.72 

6,861 

114.  2 

0. I 142 

1.25 

1.42 

65 

000 

98.7s 

7.54° 

125.8 

0.1258 

115 

1-45 

93  30 

8,360 

139.5 

O.I395 

1.05 

1 .46 

100.00 

9.125 

152.1 

0.1521 

I  03 

1-57 

100 . 00 

16,475 

174-5 

0.1745 

1 .00 

1-75 

100.00 

10,720 

178.8 
210.0 

0.1788 
0. 2100 

1 .00 

1.78 

14,620 

244.0 

0 . 2440 

0.90 

2.20 

100  .00 

TABLE  16. 
Disinfectant  Action  of  Benzoic  Acid  on  B.  coli  in  i  per  Cent  Peptone  Solution. 


Acid  Parts 
in  1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissocia- 
tion 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria 

per  c.c. 

before 

Treatment 

Percentage  Re- 
duction of  Bac- 
teria after  40 
Min. 

1,638 
1,720 
2,105 
3.365 
5.555 

13  18 
14. 11 
17.20 
27.60 

47  30 

0.0132 
0.0141 
0.0172 
0.0276 
0 . 047  ? 

6.4 
6.2 
5.8 

5-2 
3-2 

0.84 
0.87 
1 .00 
1-43 
I-5I 

70,000 

70,000 

100,000 

100,000 

100,000 

28.6 

28.6 

0.0 

730 

100. 0 

TABLE  17. 
Disinfectant  Action  of  Benzoic  Acid  on  B.  typhi  in  t  per  Cent  Peptone  Solution. 


Acid  Parts 
in   1,000,000 

Hydrogen 
Parts  in 
1,000,000 

Normality 

Per  Cent  of 
Dissocia- 
tion 

Dissociated 

Hydrogen. 

Parts  in 

1,000,000 

Bacteria 

per  c.c. 

before 

Treatment 

Percentage  Re- 
duction of  Bac- 
teria after  40 
Min. 

1.434 
1,562 
2,15s 
2,917 

11.72 
12.80 
17.80 
24.30 

0.0117 
0.0128 
0.0178 
0.0243 

6.9 
6.6 

S-7 
4-9 

0.81 
0.84 
1. 01 
I.  19 

50,000 
50,000 
s6,ooo 
56,000 

20.00 

20.00 

81.43 

100.00 

normal.  In  the  presence  of  peptone  the  required  strength  is  o.  1395 
normal.  With  B.  coli  the  corresponding  100  per  cent  reduction 
strengths  for  benzoic  acid  are  0.0199  i^  water  and  0.0473  in  peptone 
solution.  With  B.  typhi  the  respective  strengths  of  benzoic  acid  are 
0.0057  and  0.0243. 

In  a  general  way  we  may  say  that  the  presence  of  i  per  cent  pep- 
tone solution  diminishes  the  toxicity  of  hydrochloric  acid,  measured 
in  terms  of  dissociated  hydrogen,  to  from  one-eighth  to  one-tenth  its 
water  value,  and  that  of  sulphuric  acid  to  from  one-fifth  to  one-eighth 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       279 

its  water  value.  The  toxicity  of  benzoic  acid,  measured  in  normality, 
is  diminished  under  the  same  conditions  to  from  one-fourth  to  one- 
half,  and  that  of  acetic  acid  to  a  little  over  one-half  its  water  value. 

The  most  probable  explanation  of  this  phenomenon  is  the  forma- 
tion of  a  loose  compound  between  the  proteid  molecules  and  the  acids 
which  would  diminish  the  toxicity  of  the  latter,  just  as  Stiles  and  Beers 
(1905)  have  shown  that  such  a  combination  alters  the  effect  of 
mineral  salts  on  muscle. 

Bugarzky  and  Liebermann  (1898),  Cohnheim  and  Krieger  (1900), 
and  other  observers,  have  proved  the  existence  of  such  loose  com- 
pounds between  proteids  and  acids  by  freezing-point  determinations. 
We  desired,  however,  to  examine  the  actual  substance  used  in  our 
own  experiments.  Through  the  kindness  of  Dr.  Raymond  Haskell, 
of  the  Research  Laboratory  of  Physical  Chemistry  of  this  Institute 
determinations  of  electrical  conductivity  were  made  on  the  peptone 
solution  used  in  our  experiments,  on  a  solution  of  hydrochloric  acid 
in  distilled  water,  and  on  a  solution  of  the  same  strength  in  the  pep- 
tone solution. 

The  specific  conductivity  of  the  peptone  solution  was  0.000.^, 
showing  that  it  was  fairly  pure.  That  of  the  hydrochloric  acid  solution 
0.02  normal  or  720  parts  HCl  per  million,  90.46  per  cent  dissociated, 
was  0.007.  The  I  per  cent  peptone  solution  containing  0.02  normal 
hydrochloric  acid  gave  a  conductivity  of  0.002,  showing  that  approxi- 
mately four-fifths  of  the  hydrochloric  acid  had  been  neutralized  by 
the  peptone. 

It  is  evident  that  the  effect  of  the  peptone  in  decreasing  the  toxicity 
of  the  hydrochloric  acid  may  be  explained  by  the  fact  that  the  numlx-r 
of  dissociated  hvdrogen  ions  is  decreased  by  the  peptone  in  the  same 
degree.  The  effect  would  naturally  be  less  marked,  as  we  find  to  be 
the  case,  with  sulphuric  acid,  since  this  acid  is  less  ionized  to  start 
with.  Finally,  the  un-ionizcd  organic  acids  are  least  affected.  The 
decreased  toxicity  which  does  occur  with  them  may  perhaps  be  due 
to  a  loose  compound  with  their  whole-molecule — what  Stiles  and  Beers 
call  a  "physiological  compound." 

6.     GENER.\L  CONCLUSIONS. 

It  appears  from  our  experiments  that  the  typhoid  bacilkis  is  highly 
sensitive  to  an  excess  of  acid,  being  destroyed  in  an  aqueous  suspen- 


28o  C.-E.  A.  WiNSLOW   AND    E.  E.  LOCHRIDGE 

sion  by  40  minutes'  exposure  to  a  0.005  normal  solution  of  either 
hydrochloric,  sulphuric  or  benzoic  acid.  The  colon  bacillus  will 
endure  exposure,  under  similar  conditions,  to  solutions  from  two  to 
four  times  as  strong.  Ninety-nine  per  cent  of  the  bacteria  in  a 
suspension  are  killed  by  solutions  of  from  one-half  to  two-thirds 
this  strength,  the  last  few  organisms  being  especially  resistant. 

The  mineral  acids,  hydrochloric  and  sulphuric,  are  fatal  in  con- 
centrations at  which  they  are  highly  dissociated.  Their  action  runs 
parallel,  not  to  their  normal  strength,  but  to  the  number  of  free  hydro- 
gen ions  per  unit  volume.  With  the  two  organisms  tested,  both  the 
99  per  cent  and  the  100  per  cent  reductions  were  affected,  at  the  same 
concentration  of  dissociated  hydrogen,  whichever  acid  was  used. 

The  organic  acids,  acetic  and  benzoic,  are  fatal  to  the  typhoid  and 
colon  bacilli  at  a  strength  at  which  they  are  only  slightly  dissociated. 
The  effect  here  appears  to  be  due  to  the  whole-molecule  and  is  specific 
for  each  acid,  acetic  having  only  10-20  per  cent  the  toxicity  of 
benzoic. 

The  presence  of  i  per  cent  of  peptone  greatly  diminishes  the  toxic 
action  of  acids,  the  action  being  somewhat  less  marked  with  sulphuric 
acid  than  with  hydrochloric,  and  still  weaker  with  the  organic  acids. 
In  the  case  of  hydrochloric  acid  we  find  that  the  diminished  toxicity 
is  accounted  for  bv  decreased  ionization. 

It  is  evident  that  the  action  of  organic  matter  and  other  neutral  sub- 
stances in  decreasing  toxicity  greatly  complicates  the  study  of  disin- 
fectant action.  It  will  be  necessary  to  bear  this  phenomenon  in  mind 
in  considering  the  composition  of  culture  media,  since  the  apparent 
acidity,  as  determined  by  titration,  may  be  quite  different  from  the 
effective  acidity  which  influences  living  organisms.  With  the  mineral 
acids,  any  factor  which  affects  dissociation,  such  as  the  presence  of 
neutral  salts  of  the  same  anion,  will  change  the  effective  acidity. 
In  considering  the  viability  of  disease  germs  in  sewage  and  water, 
it  is  evident  that  differences  in  dilution  and  the  effect  of  inorganic 
salts,  organic  matter,  and  suspended  solids  introduce  such  complex 
factors  that  detailed  studies  of  specific  local  conditions  are  desirable. 


Effect  of  Acids  on  Typhoid  and  Colon  Bacilli       281 

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THE  INHIBITING  EFFECT  OF  CERTAIN  ORGANIC  SUB- 
STANCES UPON  THE  GERMICIDAL  ACTI(3N 
OF  COPPER   SULPHATE* 

Earlk  B.   Phklps. 

{From  the  Sanitary  Research  Laboratory  and  Sewage  Experiment  Station  of  the  ilassachusetts  InililuU 

ol    Technology.) 

INTRODUCTION. 

The  germicidal  action  of  copper  salts  dissolved  in  water  has  fre- 
quently been  found  to  depend  largely  upon  the  character  of  the  water 
itself.  Ellms  (1905)  has  pointed  out  the  intluence  of  the  hardness  and 
turbidity  of  the  water.  Johnson  and  Copeland  (1905)  found  that  in 
a  sewage  effluent,  to  which  a  large  number  of  typhoid  organisms  had 
been  added,  an  amount  of  copper  sulphate  equal  to  a  concentration  of 
20  parts  of  copper  per  million  reduced  the  number  of  bacteria  from 
1,300,000  to  600  per  cubic  centimeter  in  15  hours.  In  distilled  water, 
other  conditions  being  the  same,  the  reduction  was  from  1,300,000  to 
II.  Kraemer  (1905)  and  Basset-Smith  (1905)  have  both  shown  that 
the  toxic  action  of  copper  on  the  typhoid  organism  is  much  greater  in 
distilled  water  than  in  tap  water. 

It  is  not  difficult  to  determine  the  nature  of  the  influence  e.xerted 
by  mineral  impurities  in  the  w-ater.  Dissolved  carbonates  bring  alx)ui 
a  direct  precipitation  of  the  copper.  Even  such  insoluble  material  as 
kaolin  has  been  shown  by  Sullivan  (1905)  to  possess  the  power  of 
reacting  with  copper  salts,  in  some  cases  completely  removing  the 
copper  from  solution.  True  and  Oglevee  (1905)  have  confirmed  the 
earlier  results  of  Nageli  showing  that  adsorption  often  plays  an  im}x)r- 
tant  role,  and  that  powdered  glass  or  sand  may  destroy  in  large  meas- 
ure the  toxic  action  of  dilute  metallic  solutions. 

In  case  of  organic  impurities  the  nature  of  the  intluence  is  not 
quite  so  clear.  Direct  precipitation  of  the  copper  may  occur,  esixxially 
in  sewages.  On  the  other  hand,  the  presence  of  certain  classes  of 
organic  matter,  such  as  leaf  infusion,  has  been  shown  to  prevent  the 
precipitation  of  copper  by  alkaline  carbonates  (Ellms,  1905).     In  such 

♦Received  for  publication  March  28,  iqo6. 

283 


284  Earle  B.  Phelps 

cases  it  must  be  assumed  that  certain  organic  compounds  are  formed. 
If  such  compounds  are  found  upon  investigation  to  be  non-toxic,  or 
to  have  a  lower  toxic  value  than  the  original  copper  salt,  this  fact  may- 
throw  some  light  upon  the  nature  of  the  toxic  action  itself. 

It  was  the  purpose  of  the  present  investigation  to  study  the  germi- 
cidal action  of  copper  sulphate  upon  the  typhoid  organism  in  distilled 
water  and  in  the  presence  of  certain  organic  compounds.  Organic 
substances  were  selected  which  would  not  in  any  case  precipitate  the 
copper,  and  which,  according  to  the  chemical  evidence,  do  not  form 
any  direct  union  with  it.  This  does  not  preclude  the  formation  of  a 
"physiological  compound,"  as  defined  by  Stiles  and  Beers  (1905), 
namely,  a  compound  which  is  not  readily  detectible  by  chemical 
means,  but  which  possesses  characteristic  physiological  properties. 
For  this  purpose  dextrose  and  peptone  were  used.  The  organic 
matter  occurring  naturally  in  Boston  tap  water,  a  colored  surface 
water,  was  also  studied. 

METHODS. 

Preparation  oj  copper  sulphate. — The  copper  sulphate  used  was 
carefully  prepared  to  assure  a  pure  product.  In  particular  was  it 
desired  to  obtain  salt  free  from  ammonia,  since  there  is  reason  to  believe 
that  the  double  or  cuprammonium  salt  will  have  a  distinctly  different 
toxic  effect  from  that  of  the  simple  copper  salt.  Some  "C.  P."  cop- 
per sulphate  crystals  were  dissolved  in  water,  making  about  a  10  per 
cent  solution.  To  this  solution  a  small  amount  of  Na2C03  was  added, 
and  the  solution  was  boiled  for  some  time  to  expel  the  ammonia.  It 
was  then  acidified  and  submitted  to  electrolysis  with  a  small  current, 
about  0.05  ampere,  and  a  potential  difference  through  the  solution  of 
about  one  volt.  Copper  was  deposited  on  the  inside  of  a  platinum 
dish  which  served  as  a  cathode.  This  deposit  of  copper  was  then 
washed  with  ammonia-free  water  and  redissolved  in  dilute  sulphuric 
acid  by  reversing  the  current.  The  product  obtained  by  crystallizing 
this  solution  was  recrystallized  from  ammonia-free  water  to  free  it 
from  the  last  traces  of  acid.  It  was  dried  for  several  days  over 
calcium  chloride  and  caustic  potash. 

Preparation  oj  potassium  sulphate. — This  salt  was  prepared  by 
twice  recrystallizing  the  best  Merck  preparation  from  ammonia-free 
water. 


Effect  of  Organic  Substances  on  Copper  Sulphate   285 

Tenth-molar  (fifth-normal)  solutions  of  these  salts  were  made  up. 
For  use  i  c.c.  of  these  solutions  was  diluted  to  100  with  ammonia-free 
water.  In  the  following  tabulated  results  all  referenees  to  the  salt 
solutions  are  to  these  dilute  (N/500)  solutions. 

The  organic  compounds. — Witte's  peptone  and  Merck's  "C.  P." 
dextrose  were  used. 

The  typhoid  cultures. — The  culture  used  (Xo.  2006)  was  one  of 
those  used  in  some  earlier  work.  It  was  obtained  from  Dr.  J.  II. 
Wright,  of  the  Massachusetts  General  Hospital.  It  was  taken  from 
the  spleen  at  an  autopsy  on  May  26,  1905.  The  clinical  diagnosis 
was  typhoid  fever.  The  culture,  according  to  Wright,  gave  all  the 
ordinary  tests  for  typhoid  fever,  including  the  Widal  test. 

It  was  submitted  to  the  ordinary  diagnostic  tests,  with  the  follow- 
ing results:  fermentation  tube  with  dextrose  broth,  growth  in  closed 
arm,  acid  produced,  no  gas ;  milk  not  coagulated,  slight  acid  production ; 
no  indol  from  peptone;  nitrates  reduced  slightly;  gelatin  not  liquefied 
in  two  weeks;  growth  on  agar  slant,  scanty,  thin,  translucent;  it 
reacts  with  the  sera  of  two  rabbits  into  which  two  other  strains  of 
typhoid  organisms  had  been  introduced  subcutaneously. 

Cultures  A,  B,  and  C  w^re  obtained  from  plates  made  from 
Bottle  2  in  Experiment  5.  They  had  lived  for  48  hours  in  the 
presence  of  a  concentration  of  0.63  parts  of  copper  per  million  in 
distilled  water.  They  gave  all  the  above  tests  exactly  as  the  original 
culture,  except  that  they  were  not  submitted  to  the  Widal  test. 

Technique  0}  the  experiments. — The  experiments  were  made 
under  as  uniform  conditions  as  possible,  and  in  the  following  manner: 

In  all  cases,  except  where  tap  water  is  specified,  sterilized  ammonia- 
free  water  was  used.  When  dextrose  or  other  solutions  were  used, 
these  were  made  up  with  ammonia-free  water  and  sterilized.  The 
organism  was  grown  for  24  hours  at  37°  on  the  surface  of  an  agar 
slant.  A  loop  of  the  culture  was  then  removed  and  placcxl  in  100  c.c. 
of  water.  After  thorough  shaking,  proper  amounts  of  this  suspension 
were  added  to  the  bottles  of  water  in  which  the  experiments  were  to 
to  be  made.  These  bottles  were  then  thoroughly  shaken,  and  the 
the  bacteria  determined  in  each  by  properly  diluting  i  c.c.  and 
plating.  For  plating  agar  was  used,  and  all  plates  were  grown  for  1 5 
hours  at  37°  C.    The  salt  solution  was  then  added.    In  the  same  manner 


286 


Earle  B.  Phelps 


determinations  of  the  numbers  of  organisms  present  were  made  at 
intervals  during  the  experiment,  as  indicated  in  the  tabulated  results. 
In  the  following  tables  the  full  details  of  each  experiment  are 
first  given,  including  the  actual  bacterial  counts.  In  order  to  compare 
the  various  experiments,  the  bacterial  results  have  been  calculated 

EXPERIMENT  i. 
Started  January  24,  1906. 


Bottle 

Water 

CUSO4 

K,SO, 

No. 

(c.c.) 

Sol.  (c.c.) 

Sol.  (c.c.) 

I 

08 

0.6 

2 

98 

1 . 0 

3 

100 

2.0 

4 

08 

0.6 

5 

98 

1 . 0 

6 

99 

2.0 

Concent,  of 
Salt 

(N/ 1, 000,000) 


12 

20 
40 
12 

20 
40 


38 
63 
26 


Counts 


Initial 


380,000 
400,000 
350,000 
370,000 
410,000 
400,000 


5  Min. 


125,000 
45,000 
25,000 


64  Hrs. 


6,000 

1,700 

300 

370,000 

460,000 

400,000 


24 


Hrs. 


42 

12 

9 

50,000 
45.000 
43.000 


Temperature,  2o°-22'^ 


EXPERIMENT  2. 
Tap  Water — Started  January  29,  1906. 


6 

0 

u 

^ 

j>2 

Concent,  of 
Salt 

(N/i, 000,000) 

3 

Counts 

V 

1 

u 

Initial 

I  Min. 

10  Min. 

30  Min. 

6  Hrs. 

29  Hrs. 

I 

2 
3 
4 
5 
6 
7 
8 

9 
10 

89 

04 
84 
90 
88 
88 
89 
90 
95 
92 

0 
0 

I 

2 

3 

4 
8 
2 
0 
0 

0.4 
0.8 
1.2 
2. 0 
30 

8.8 
16.8 
28.0 

44   0 
66.0 
8.8 
18.0 
26.0 
42.0 
66.0 

0 
0 
0 

I 

2 

28 

^3 
88 

39 
08 

170,000 
180.000 
190,000 
150,000 
180,000 
140,000 
160,000 
180,000 
170,000 
200,000 

180,000 
120,000 

170,000 
100 

170,000 
100 

110,000 
so 

SO 

9,800 

510 

100 

80 

0 

240,000 

336,000 

92,000 

69,000 

80,000 

Temperature,  20°-23'^ 


EXPERIMENT  3. 
Started  February  6,  1906. 


BotUe 

Water 
(c.c.) 

CuSO, 

K,SO< 

Concent,  of 
Salt 

(N/i, 000,000) 

Copper 

(Parts  per 
Mill.) 

Counts 

No. 

Sol.  (c.c.) 

Sol.  (c.c.) 

Initial 

I  Hr. 

6  Hrs. 

24  Hrs. 

I 
2 
3 
4 
5 
6 

7 
8 

100 
99 

'9! 
99 
97 
98 
98 

0 

I 
2 
3 

5 
0 
0 
0 

0 

1 
2 
3 

5 
0 
0 
0 

10 
20 
40 
60 
10 
20 
40 
60 

0.31 
0.62 
1.24 
1.86 

125,0  jO 
135.000 
140,000 
128,000 
140,000 
136,000 
132,000 
1 40,000 

80,000 

5.300 

700 

400 

130 

1 20,000 

100,000 

100,000 

120,000 

8 
6 
9 

0 
80,000 
70,000 
63,000 
56,000 

Temperature,  2o°-2  2'^ 


Effect  of  Organic  Substances  on  Copper  Sulphate    287 


into  percentage  of  the  initial  numbers,  and  these  resuUs  have  Ixtn 
used  in  the  discussion  of  the  table. 

In  each  case  in  which  copper  sulphate  was  used  the  control  con- 
tained an  equimolar  amount  of  potassium  sulphate.  The  controls 
therefore  differed  from  the  tests  simply  in  the  substitution  of  potas- 
sium for  the  copper. 

EXPERIMENT  4. 
Started  February  13,  1006. 


d 
2; 

Water 

Q 

■•  6 

Concent,  of 

5^§ 

Counts 

V 

1 

n 

2 

0 
u 

0  6 

u 

n 

Salt 
(N/ 1, 000,000) 

Initial 

3Hrs. 

6Hr8. 

33  Hm. 

48  Hn>. 

I 

Best 

98 

o.S 

10 

o.3> 

540,000 

1,800 

1,400 

660 

660 

3 

** 

99 

1 .0 

20 

0.63 

540,000 

400 

100 

46 

350 

.s 

99 

I 

OS 

10 

0.31 

560,000 

415,000 

640,000 

190,000 

4.300 

4 

98 

I 

1 .0 

20 

0.63 

500,000 

170,000 

83,000 

100 

600 

s 

Tap 

90 

0-5 

10 

0  3« 

540,000 

I34.003 

3,000 

lii 

0 

6 

** 

87 

I.O 

20 

0.63 

560,000 

6,300 

700 

«s 

3 

7 

Best 

98 

1.0 

20 

500,000 

530,000 

95. 000 

10,000 

8 

*' 

98 

I 

1.0 

20 

500,000 

490,000 

300,000 

430,000 

9 

Tap 

90 

1.0 

20 

.... 

540,000 

600,000 

110,000 

l.JOO 

Temperature,  2o°-24°, 

"Best  water"  is  ammonia-free,  distilled  water. 

EXPERIMENT  5. 
Started  Febrdary  15,  1906. 


Bottle 

Water 

(c.c.) 

Dextrose 
(gm.) 

CuSo, 
Sol. 
(c.c.) 

K,So4 
Sol. 
(c.c.) 

Concent,  of 

Salt, 

(N' 1,000,000) 

Copper 

(Parts  per 

MiU.y 

Cor  NTS 

No. 

Initial 

6Hrs. 

34  Hrs.     48  Hrs. 

I 
2 
3 
4 
5 
6 

7 
8 

100 
100 
99 
100 
98 
99 
99 
98 

I 
I 

I 
I 

o.S 
1 .0 

0.5 
1.0 

OS 
1.0 

o.S 
1.0 

10 
20 
10 
20 
10 
30 
10 
20 

0.31 
0.63 

o.3> 
0.63 

30,000 
26,000 
26,000 
38,000 
38,000 
30.000 
34,000 
24,000 

600 
300 
1,300 
conum 
30.000 
37,000 
35,000 

3I,000 

184 

70 

1.800 

inated 

14.000 

10,000 

13,000 

13,000 

40 

23 

900 

9.000 

8,000 

Temperature,  22°-24°. 


EXPERIMENT  6. 
Started  February  20,  1906. 


CuSO« 

K,S04 

Concent,  of 

Copper 

COOKTS 

Bottle 

Water 

Sol. 

Sol. 

Culture 

Salt 

(Parts  per 
Mill.) 

No. 

(c.c.) 

(c.c.) 

(c.c.) 

(N/ 1,000,000) 

Initial 

34  Hr,. 

1 

98 

I 

2,006 

30 

0.63 

44,000 

460 

2 

98 

1 

" 

20 

50,009 

40.000 

3 

90 

I 

A 

20 

0.63 

69.000 

600 

4 

98 

I 

*' 

20 

90,000 

44-000 

5 

98 

I 

B 

20 

0.63 

4^-000 

400 

6 

97 

I 

•' 

20 

.... 

48,.XX> 

36,000 

7 

98 

I 

C 

20 

0.63 

61,000 

boo 

8 

98 

I 

20 

67,000 

25.000 

Temperature,  21-23' 


288 


Earle  B.  Phelps 


EXPERIMENT  7. 
Started  March  5,  1906. 


Bottle 

Water 

Pepton 

(gm.) 

Culture 

CUSO4 
Sol. 

(c.c.) 

Concentration 
of  Salt 

(N/ 1,000,000) 

Copper  (Parts 

Counts 

No. 

(c.c.) 

No. 

per  Mill.) 

Initial 

24  Hrs. 

I 

100 

0 

2,006 

0.5 

10 

0.31 

179,000 

1,040 

2 

100 

0 

" 

2 

0 

40 

1.24 

189,000 

3 

3 

lOI 

0 

A 

0 

5 

10 

0.31 

2,400 

3 

4 

100 

0 

" 

2 

0 

40 

1.24 

2,200 

0 

S 

98 

I 

2,006 

0 

5 

10 

0  31 

171,000 

2,420,000 

6 

lOI 

I 

2 

0 

40 

1.24 

175.000 

455.000 

7 

9Q 

I 

A 

0 

5 

10 

0-31 

2,800 

19,500 

8 

98 

I 

2.0 

40 

1.24 

2,900 

15,000 

Temperature,  20°-24°. 

DISCUSSION   OF   RESULTS. 

Effect  of  organic  matter  in  tap  water. — In  comparing  the  results 
of  these  experiments,  the  basis  of  comparison  will  be  the  number  of 
surviving  organisms  in  each  case,  expressed  as  percentage  of  the 
initial  number.  Other  bases  of  comparison  suggest  themselves,  but 
the  one  selected  appears  on  the  whole  to  be  the  most  logical.  In 
this  way  the  effect  of  the  copper  upon  those  few  comparatively  re- 
sistant organisms,  always  found  in  experiments  of  this  nature,  is 
given  predominant  importance,  while  the  effect  upon  that  large  per- 
centage of  organisms  which  is  killed  in  all  the  experiments  has  com- 
paratively little  weight. 

Such  a  comparison  of  the  average  results  of  Experiments  i  and 
3,  best  water,  with  those  of  Experiment  2,  tap  water,  gives  the 
following  figures: 


Concentration  of 

Per  Cent  Surviving— 24  Hrs. 

Ratio 

(6-a) 

Copper 

(iV/'i,ooo,ooo) 

Best  Water 

(a) 

Tap  Water 
(b) 

10 
20 
40 
60 

0.009 
0.004 
0.004 
0.000 

5.  800 
0.  280 
0.053 
0.000 

644 
70 
13 

It  is  quite  evident  that  the  organic  matter  present  in  the  tap  water 
inhibits  the  toxic  action  of  the  copper,  but  the  significant  fact  here  is 
that  there  is  no  definite  point  in  the  series  at  which  the  addition  of 
more  copper  to  the  tap  water  will  produce  a  normal  killing  effect 
such  as  is  produced  in  pure  water.     The  effect  of  the  organic  matter 


Effect  of  Organic  Substances  on  Copper  Sulphate    289 

cannot  be  neutralized  up  to  the  concentration  recjuired  for  the  com- 
plete elimination  of  the  organisms.  It  would  appear,  therefore,  that 
the  apparent  inhibition  of  the  toxic  efTect  of  the  copper  is  due  to  the 
formation  of  some  non-toxic  compound;  that  the  formation  of  this 
compound  is  due  to  a  reversible  reaction  which  is  complete  only  at 
the  highest  concentration  of  copper  used;  and  that  Ix-low  this  point 
the  addition  of  increasing  amounts  of  copper  sim{)ly  brings  aljout  a 
further  reaction  toward  this  non-toxic  compound,  producing  a  new 
condition  of  equilibrium  according  to  the  law  of  mass  action. 

Dextrose. — The  effect  of  dextrose  upon  the  coi)per  sulphate  seems 
to  be  of  a  quite  different  nature.  The  following  figures  are  calcu- 
lated from  the  results  of  Experiment  4 : 


Concentration  of 

Copper 

(N,  1 ,000,000) 

Per  Cent  Surviving — 24  Hrs. 

Without 
Dextrose 

(<J) 

With  Dextrose 
(fr) 

Ratio 
(a  +  b) 

10 
20 

0. 12 
0.009 

34  0 
0.02 

283.0 
2.2 

In  the  lower  concentration  dextrose  neutralizes  completely  the 
toxicity  of  the  copper,  so  that  the  effect  is  about  equal  to  that  in  the 
control  (Bottle  8).  In  the  higher  concentration  the  effect  of  the 
dextrose  is  almost  nil.  This  result,  taken  in  connection  with  the  fact 
that  the  concentration  of  the  dextrose  is  about  -jV  molar,  and  that 
there  are  accordingly  over  10,000  C^H.jOo  molecules  to  each  copper 
ion  in  the  one  case  and  over  5,000  in  the  other,  leads  to  the  con- 
clusion that  the  results  obtained  are  not  due  to  the  formation  of  a 
non-toxic  compound.  A  careful  study  of  the  electric  conductivity  of 
these  solutions  did  not  reveal  any  decrease  in  the  normal  conductivity 
of  the  copper  due  to  the  presence  of  dextrose.  It  may  be  presumed, 
however,  that  what  has  been  called  a  "physiological  compouml" 
might  be  readily  broken  down  under  the  inlluence  of  the  electric 
current. 

The  following  assumption  seems  to  be  in  agreement  with  all  the 
facts:  The  bacteria  doubtless  attract  to  themselves  by  a  process  of 
adsorption  a  certain  amount  of  dextrose,  and  are  thus  surrounded 
by  a  solution   of  this  sugar  more  concentrated  than  that  existing  in 


290  Earle  B.  Phelps 

the  water.  This  tends  to  produce  a  difference  in  osmotic  pressure 
between  the  free  solution  and  this  surrounding  layer,  and  it  may  be 
that  a  certain  definite  concentration  of  the  copper  ions  is  necessary 
before  this  film  of  "osmotic  tension"  can  be  pierced.  An  analogous 
case  is  that  of  surface  tension,  which  presents  a  certain  resistance 
to  the  entrance  of  a  non- wetted  body. 

Peptone. — Peptone  was  the  third  substance  studied.  The  results 
of  Experiment  7  show  that  a  i  per  cent  solution  of  peptone  allows  the 
typhoid  organism  to  multiply  even  in  the  presence  of  rather  strong 
copper-sulphate  solution.  Owing  to  the  high  electric  conductivity 
of  the  rather  impure  peptone,  the  results  of  conductivity  determina- 
tions are  uncertain.  There  seems  to  be  little  doubt  here,  however, 
that  an  actual  combination  has  taken  place  between  the  copper  and 
the  peptone.  The  addition  of  sufficient  copper  solution  to  the  peptone 
solution  to  give  a  distinct  color  resulted  in  the  formation  of  a  colloidal- 
like  solution,  of  a  robin's-egg  blue  color. 

These  results  have  an  important  bearing  upon  many  practical 
points  in  connection  with  the  use  of  copper  sulphate  for  the  destruc- 
tion of  the  typhoid  organism.  Results  obtained  in  the  laboratory 
in  distilled  water  are  not  in  the  least  indicative  of  what  may  be 
expected  under  field  conditions.  Neither  are  actual  field  results 
on  one  water  reliable  criteria  for  the  undertaking  of  similar  work 
upon  another.  The  impracticability  of  the  internal  use  of  copper 
sulphate  in  the  treatment  of  typhoid  fever  is  also  suggested  by  the 
results  obtained  with  peptone. 

Selection  0}  a  resistant  strain. — Cultures  A,  B,  and  C  were  taken 
from  Bottle  2  in  Experiment  5,  and  had  lived  for  48  hours  in  a  solu- 
tion of  copper  sulphate  containing  0.63  parts  of  copper  per  million. 
They  were  used  in  Experiments  5  and  6  in  parallel  with  the  original 
strain  in  order  to  determine  whether  they  possessed  any  increased  resist- 
ance to  copper.  The  results  are  all  negative,  indicating  that  these 
strains  are  not  more  resistant  than  is  the  parent  strain. 

The  time  factor  in  the  germicidal  effect. — In  Experiments  i,  3,  and 
4,  where  determinations  of  the  numbers  of  organisms  were  made 
at  various  intervals  during  the  experiment,  it  is  seen  that  even  in  the 
more  dilute  solution  used  there  is  a  very  rapid  falling-off  in  numbers 
during  the  earlier  part  of  the  test.     In  Experiment  i,  for  instance. 


Effect  of  Organic  Substances  on  Copper  Sl'lphate    2qi 

the  reduction  in  5  minutes  was  67  per  cent  in  a  concentration  of  o..^8 
parts  of  copper  per  million,  but  all  the  organisms  had  not  been  killed 
in  24  hours.  Such  results  indicate  an  extreme  range  of  resistance 
to  copper  among  these  organisms.  The  42  organisms  which  sur- 
vived the  treatment  for  24  hours  lived  288  times  as  long  as  the  250,- 
000  which  died  in  less  than  5  minutes. 

Ejject  of  concentration. — This  great  variability  is  also  shown  in  a 
study  of  the  effect  of  various  concentrations. 

The  average  percentage  survival  after  24  hours  in  all  experi- 
ments with  0.38  parts  of  copper  jx-r  million  was  o.ig;  with  0.76 
parts,  0.07;  with  1.26  parts,  0.04.  Notwithstanding  the  fact  that 
99.8  per  cent  are  killed  by  a  concentration  of  0.38  parts,  there 
are  still  0.04  per  cent  which  can  withstand  the  action  of  a  copper 
solution  four  times  as  concentrated. 

This  tremendous  variability  is  of  vital  importance,  not  only  in 
questions  of  sterilization  of  water  by  heat,  freezing,  or  chemicals,  in 
all  of  which  cases  such  a  variability  has  been  shown  to  exist,  but 
in  the  far  more  important  questions  of  the  self-purification  of  streams 
and  the  longevity  of  the  typhoid  bacillus  in  the  water.  Most  cases 
of  typhoid  fever,  contracted  from  drinking-water,  have  undoubtedly 
come  from  these  few  resistant  forms  rather  than  from  those  which 
are  known  to  have  perished  in  the  natural  stream.  \  removal 
of  99.99  per  cent  of  the  typhoid  organisms  may  sound  like  security 
but  actually  means  high  typhoid  rates.  The  importance  of  this 
residual  hundredth  of  a  per  cent  cannot  be  overestimated,  owing  to 
the  very  fact  that  it  is  so  resistant. 

REFERENCES. 

Bassett-Smith,  p.  W.     Jour.  Prev.  Med.,  1905,  13,  p.  388. 

Ellms,  J.  W.     Jour.  N.  E.  W.  W.  A.,  1905,  19,  p.  496. 

Johnson,  C.  A.,  and  Copeland,  W.  K.    Jour.  Inject.  Dis.,  1905,  Suppl.  No.  i.  p.  327. 

Kraemer,  H.     Proc.  Amer.  Phil.  Soc,   1905,  49,  p.  51. 

Stiles,  P.  G.,  and  Beers,  W.  H.,  Jr.    Am.  Jour.  0}  Physiol.,  1905,  14,  p.  133. 

True  and  Oglevee.     Bot.  Gaz.  1905,  39,  p.  i. 

Sullivan,  E.  C.     Jour.  Am.  Chem.  Soc,  1905,  27,  p.  976. 


A  NEW  SOLUTION  FOR  THE  PRESUMPTIVE  TEST 
FOR  BACILLUS  COLL* 

Daniel  D.  Jackson. 

{From  Ml.  Prospect  Laboratory,  Brooklyn,  N.  F.) 

MacConkey  "•'•'  has  pointed  out  that  the  intestinal  bacteria  grow 
well  on  an  agar  medium  containing  o .  5  per  cent  of  sodium  tauro- 
cholate,  while  the  common  bacteria  are  to  a  great  extent  excluded, 
and  he  has  recommended  that  this  medium  be  used  to  distinguish 
between  B.  coll  and  B.  typhi  ahdominalis  and  also  as  a  test  for  fecal 
contamination  in  water.  Jordan,  Russell,  and  Zeit,-*  in  experiment- 
ing with  this  medium  for  testing  water,  did  not  obtain  very  favorable 
results  in  that  the  medium  failed  to  eliminate  all  of  the  water  forms. 
Robin^  has  used  MacConkey's  agar  to  advantage  in  tests  of  filters; 
but  while  the  results  were  preferable  to  those  obtained  on  the  regular 
agar,  it  could  not  be  concluded  that  all  the  bacteria  found,  or  a  definite 
percentage  of  them,  were  always  of  fecal  origin. 

In  order  to  determine  the  germicidal  action  of  the  bile  salts  and 
their  constituents,  the  author  has  used  a  standard  agar  medium  with 
an  addition  of  varying  amounts  of  these  salts,  and  noted  the  effect 
produced.  The  following  are  the  results  obtained  with  the  bile  salts, 
and  their  constituents  upon  the  agar  growth  at  37°  C.  of  bacteria  from 
a  slightly  contaminated  water  containing  two  B.  coli  per  c.c. 

TABLE  I. 


Sodium  taurocholate 

"       glycocholate 

Taurocholic  acid.  .  . 

Glycocholic  add. . . 

Cholic  acid 

Taurin 

Glycin 


Plain  Agar 


20 
20 
20 
20 
20 
20 
20 


Agar  +0.05% 


14 
14 
10 
12 

14 
20 
19 


Agar +0.5% 


4 
2 
2 

S 
2 

19 
II 


Agar  +  2.5% 


2 

2 
o 
o 
o 

20 

s 


This  table  shows  that  the  bile  acids  reduced  the  number  of  bac- 
teria even  when  only  o .  05  per  cent  was  taken,  and  that  when  o .  5 
per  cent  was  employed,  taurocholic,  gylcocholic,  and  cholic  acids, 
sodium  taurocholate,  sodium  glycocholate,  and  glycin  greatly  reduce 
the  bacteria  present,  some  of  those  remaining  giving  the  tests  for 

*  Received  for  publication  April  12,  1906. 

292 


Presumptive  Test  for  Bacillus  Coli 


293 


B.  coli.  When  2.5  per  cent  of  the  sails  were  used  ihe  bacieriu  were 
killed  by  the  bile  acids  and  considerably  reduced  by  j^lycin,  whik-  the 
results  with  the  bile  salts  remained  about  the  same. 

It  is  evident  that  the  bile  salts  and  especially  their  acids  exert  a 
strong  restraining  action  on  the  common  bacteria  which  grow  at  blood 
heat,  and  that  except  for  glycin  (which  mildly  restrains  much  as  a  sugar 
retards  bacterial  action),  the  actual  bactericidal  effect  lies  in  the 
cholic  acid  radical  of  these  salts. 

It  is  evident,  therefore,  that  either  of  the  bile  sails  or  a  mixture  of 
both  salts  may  be  employed,  thus  greatly  reducing  the  cost  and  ease 
of  preparation  of  what  would  otherwise  be  a  medium  imjjracticable 
for  use  in  laboratories  where  much  water  work  is  carried  on.  In  fact 
it  allows  of  the  use  of  plain  bile  as  a  liquid  restraining  medium. 

In  order  to  determine  whether  or  not  this  restraining  action  is  selec- 
tive, a  series  of  waters  and  solutions  were  made  containing  varj-ing 
numbers  of  intestinal  bacteria.  The  following  table  gives  the  results 
obtained : 

TABLE  2. 
Comparison  of  Results  from  Various  Bii.e  Agar  Media. 


Gelatin  at  20°  C 

Agar  at  38°  C 

Bile  agar  (fresh  ox  bile  and  agar) 

Bile  agar  (with  bile  diluted  i-i) 

Lartosc  bile  agar  (with  bile  diluted   i-i) 

Litmus  lactose  bile  agar  (with  bile  diluted  i-i) 


Bactebia  per  c.c. 


Uncontami- 
nated  Shal- 
low  Dug 
Well 


930 

25 
o 

U 
o 
o 


Contami- 
nated I'ond 
Water 


2700 
170 
j6 
43 
25 
17 


Suspension 

of  Feces 

rronously 

Grown  at 

37°  C. 


350,000 
450,000 
bo.ooo 
300,000 
350,000 
350,000 


Frrsh 

Susprn<unn 

ContaininK 

B.  coli. 


000,000 

000,000 
000,000 
000.000 
675,000 
600,000 


The  media  used  in  the  experiments  shown  in  the  foregoing  table 
were  made  from  fresh  ox  bile  instead  of  the  usual  meat  infusion  with 
peptone.   The  best  results  were  obtained  when  the  bile  was  not  diluted. 

The  figures  show  that  a  strong  restraining  action  is  e.xerted  against 
the  growth  of  the  common  bacterial  flora  and  to  little  or  no  extent 
against  certain  of  the  fecal  bacteria. 

The  test  for  fecal  bacteria  is  an  important  matter,  esix-cially  in 
water  analysis.  The  method  generally  employed  at  the  present  time 
for  routine  work  was  devised  by  Dr.  Theobald  Smith,'*   and  con- 


294  Daniel  D.  Jackson 

sists  of  obtaining  the  percentage  of  gas  formed  by  bacterial  growth 
in  the  closed  arm  of  an  inverted  tube  containing  a  solution  of  meat 
extract,  peptone  and  lactose  or  dextrose.  One-tenth,  i,'and  lo  c.c.  of 
water  are  added  to  the  sterilized  media  and  allowed  to  stand  48  hours 
in  an  incubator  at  37.5° C.  At  the  end  of  this  time  the  percentage 
of  gas  formed  and  the  amount  absorbed  by  caustic  is  obtained. 

Typical  growths  of  B.  coli  give  from  25  to  70  per  cent  gas,  of  which 
from  25  to  40  per  cent  is  carbonic  acid  and  is  absorbed  by  caustic. 
This  so-called  "presumptive  test"  is  used  extensively  in  most  water 
laboratories,  and  in  those  of  the  New  York  City  Department  of  Water 
Supply,  Gas,  and  Electricity  there  are  tested  annually  in  this  manner 
about  7,000  samples  of  water.  The  difficulty  with  this  test  lies  in 
the  fact  that  when  badly  contaminated  water  is  taken  as,  for  instance, 
a  suspension  of  feces  or  a  strong  sew'age,  the  test  often  utterly  fails, 
due  to  an  overgrowth  of  some  germ  other  than  B.  coli. 

In  such  cases  B.  coli  is  inhibited  in  its  growth  by  the  products  of 
metabolism  of  certain  other  species,  usually  streptococci.  This  inhi- 
bition has  been  noticed  by  Prescott  and  Baker,^  Irons,^°  and  others, 
and  has  been  a  constant  source  of  annoyance  to  the  author  in  work 
on  water  and  sewage.  The  use  of  sodium  taurocholate  as  suggested 
by  MacConkey  to  prevent  the  growth  of  these  inhibiting  bacteria  is 
a  step  in  the  right  direction,  but  the  salt  is  very  expensive  and  difficult 
to  obtain,  and  the  amount  suggested  (0.5  per  cent),  while  effective 
in  soHd  media,  is,  in  the  opinion  of  the  author,  too  small  for  liquid 
media. 

Inasmuch  as  the  foregoing  experiments  showed  an  equal  effective- 
ness of  the  more  abundant  sodium  glycocholate  there  appeared  to  be 
no  reason  why  a  mixture  of  the  two  bile  salts  (Platner's  crystallized 
bile)  could  not  be  used.  This  mixture  is  easy  to  obtain  and  cheap 
enough  to  use  with  liquid  media  in  larger  amounts  than  those  em- 
ployed by  MacConkey.     It  is  made  as  follows: 

Concentrate  ox  bile  to  one-fourth  its  bulk;  mix  with  animal  charcoal  in  a  mortar 
to  a  thick  paste  and  evaporate  to  complete  dryness  over  a  water  bath. 

To  the  dry  charcoal  bile  mixture  add  five  volumes  of  absolute  alcohol.  Shake 
from  time  to  time,  and  in  about  half  an  hour  filter  off  the  alcohol.  Concentrate 
the  filtrate  by  boiling;  and  when  cold  add  ether  in  large  excess  and  the  crystallized 
sodium  salts  of  taurocholic  and  glycocholic  acid  will  be  precipitated.  Allow  the 
precipitate  to  stand  over  night  in  a  tightly  covered  glass  jar  and  decant  off  the  mix- 
ture of  alcohol  and  ether. 


Presumptive  Test  for  Bacillus  Coli 


295 


Dry  the  precipitated  salts  first  on  the  water  bath  and  then  in  an  oven  kept  at  100" 
C.  Powder  in  a  mortar  and  keep  in  a  tightly  stoppered,  wide  mouth  glass  bottle. 
Purify  if  necessary,  by  redissolving  in  a  small  quantity  of  warm  alcohol  and  again  pn-- 
cipitating  with  a  large  excess  of  ether. 

The  author's  experiments  with  bile  agar  suggested  the  use  oi  bile 
itself  with  i  per  cent  of  lactose  as  a  liquid  to  rc-i)lace  Smith  solution, 
thus  making  a  much  more  effective  medium  which  would  be  cheaper 
and  easier  to  prepare  than  the  latter  solution.  In  the  following  experi- 
ments the  results  with  the  new  bile  lactose  medium  is  compared  with 
plain  Smith  solution,  Smith  .solution  containing  .sodium  taurochi.late, 
and  the  same  solution  containing  sodium  glycocholate. 

It  is  evident  that  the  presence  of  either  or  both  of  the  bile  .sali.>, 
favors  the  growth  of  B.  coli  in  Smith  solution  by  inhibiting  the  growth 
of  other  bacteria.  A  proper  medium,  may,  therefore,  be  made  by 
adding  to  Smith  solution   9   per  cent    of   Platner's  crystallized   bile 

TABLE  3. 


Test  on  Sewage 


Total  Gas 


.\fter  Absorption 


%  Carbonic  Add 


Teal  for   B.  coli 


Smith  solution- 
o.  I  c.c. ... 
1.0       .... 


10 
Smith 


10 
Smith 


o 
I 

10 

Bile 

o 

I 

10 


and  taurocholate 

I  c.c 

o       


and  glycocholate — 


I  c.c. 
o 
o 

lactose 
I  c.c. 
o 
o 


12 
12 
17 

S2 
43 
6S 

33 
26 

47 

25 
3S 
27 


33 
27 
40 

33 
18 
3» 

18 
35 
19 


37 
37 
38 

30 
JI 
34 

28 
JO 


o 
o 
o 

+ 
+ 
+ 

+ 

+ 


Test  on  Solution  of  Horse 
Feces 

Totid  Gas 

After  Absorption 

%  Carlx)nic  .\cid 

Test  for  B.  (Mi 

Smith  solution — 

0. 1    c.c 

0 
0 
0 

0 

35 

34 

33 
18 
26 

2S 

40 
67 

0 
0 
0 

0 
18 
23 

»3 

18 

«7 
28 
41 

0 
0 
0 

0 

M 

30 

M 

S> 
30 
30 

0 

1 .0        

0 

lo.o         

0 

Smith  and   taurocholate — 
0. 1  c.c.  ... 

0 

1 .0       

+ 

to.o       

+ 

Smith  and  glycocholate — 
0. 1  c.c 

+ 

1 .0       

0 

10.0        

+ 

Bile  lactose — 

0. 1  c.c 

+ 

1.0         

4- 

+ 

296 


Daniel  D.  Jackson 


prepared  as  previously  described,  or  by  using  the  author's  bile  lactose 
medium. 

This  latter  medium  is  to  be  preferred  when  fresh  bile  can  be 
obtained,  as  it  appears  to  be  somewhat  more  effective  and  is  cheaper 
and  easier  to  prepare.  Several  brands  of  inspissated  bile  were  tried 
but  they  were  all  very  acid  and  even  when  neutralized  were  not  effec- 
tive.    The  bile  lactose  medium  is  prepared  in  the  following  manner: 

Fresh  ox  bile  from  the  slaughter  house  is  poured  into  flasks  and  sterilized  20  min- 
utes under  15  pounds  pressure.  When  ready  for  use  the  bile  is  fi,ltered  and  i  per  cent 
lactose  is  added.  It  is  then  poured  into  Smith  tubes  and  again  sterilized.  If  the  bile 
is  fresh  the  acidity  will  be  from  zero  to  5  by  Fuller's  scale,  and  within  these  limits 
there  is  no  interference  with  the  results. 

A  large  number  of  tests  for  B.  coli  were  carried  on  to  determine 
the  relative  efficiency  of  bile  media  as  compared  with  Smith's  solu- 
tion, and  also  to  show  the  selective  action  of  the  bile  salts.  Some  of 
the  most  conclusive  results  are  given  in  Tables  3  and  4. 

TABI.E  4- 
Comparison  of  Results  Obtained  in  the  Test  for  B.  Coli  with  Smith  Solution  and  with 

Lactose  Bile. 


Solution  No.   i   containing  pure 

culture  B.  Coli — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Solution  No.   2   containing  pure 

culture  B.  Coli — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Pond     water,  slightly    contami- 
nated— 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Pond  water ,  contaminated — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

New   York  Bay  water,  contami- 
nated. No.  I — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

New  York   Bay  water,  contaroi 

nated,  No.  2 — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 


Smith  Solution 


o. I  c.c. 


36 
22 
38 

-I- 


30 
20 

33 


25 
18 
28 


26 
20 

23 
o 


37 

23 

40 


27 
19 
30 

-f 


17 


40 
25 
28 


60 

45 
25 


36 
24 
33 


72 
40 

44 
+ 


13 


Bile  Lactose 


32 
20 
37 

+ 


49 

33 
33 


43 
22 
49 


29 
16 

45 

-I- 


I  C.C. 


46 

28 

39 


6s 
45 
26 

-I- 


o 
o 
o 
o 

41 

23 

44 


35 
19 
46 


35 
22 

37 

+ 


10  C.c. 


45 
27 
40 


53 
26 

34 

+ 


33 
21 
36 


52 
29 
45 


57 
32 
44 


44 
25 
43 
-I- 


Presumptive  Test  for  Bacillus  Coli 

TABLE  4.— Continued. 


297 


Smith  Solution 


0.1  c.c. 


Rahway  River,  N.   J.,   just    be- 
low sewer  outlet — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Bodine     Creek,    S.     I.,    badly 
contaminated — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Kill  von  KuU,    badly   contami- 
nated— 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Hudson  River  at  mouth,  badly 
contaminated — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Hudson     River      at     Hoboken, 
badly  contaminated — 

TotaJ  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

East  River  near   B'klyn  Bridge. 
badlv  contaminated — 

Total  gas 

After  absorption.  .  . ._ 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

East    River     below    Blackwell's 
Island,  contaminated — 

Total  gas 

Alter  absorption. 

Per  cent  carbonic  acid 

Test  for  B-  Coli 

Harlem  River  near  mouth,  con- 
taminated— 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Bronx  River,  contaminated — 

Total  gas 

After  absorption  .  .  .  ._ 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Gowanus  Canal,  at  head,  badly 

contaminated — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid .... 

Test  for  B.  Coli 

Gowanus  Canal  at  Hamilton  Av- 
enue bridge,   badly    contarai 

nated — 

Total  gas 

After  absorption  . . .  ._ 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Sewage  effluent  after  rough  fil- 
tration, Bedford,  N.  Y. — 

Total  gas 

After  absorption 

Per  Cent  carbonic  acid 

Test  for  B.  Coli 


17 


15 


25 
17 
3a 


33 
23 
30 


J5 


I  c.c. 


13 


37 
25 
37 

-t- 


41 

30 
26 

-I- 


19 


o 

23 


13 


J7 


10   C.c. 


36 
25 
27 


13 


13 


40 

27 

33 


23 


17 


18 


Bile  Lactose 

O.I  C.C. 

I  C.c. 

10  C.C. 

50 

20 

30 

28 

"7 

18 

44 

41 

40 

-t- 

-»- 

+ 

53 

32 

6s 

30 

23 

43 

40 

38 

38 

+ 

+ 

+ 

35 

32 

48 

22 

M 

33 

3> 

31 

-t- 

+ 

-f 

32 

a 

35 

18 

«7 

lb 

44 

SI 

36 

-t- 

-^ 

+ 

37 

30 

4a 

17 

— 

a? 

37 

— 

36 

+ 

0 

-♦- 

51 

34 

40 

36 

25 

a6 

49 

a6 

30 

-t- 

+ 

-h 

25 

27 

3S 

>7 

17 

ao 

32 

37 

43 

-t- 

-♦- 

+ 

27 

36 

as 

10 

10 

16 

30 

27 

36 

+ 

-H 

+ 

33 

50 

33 

23 

30 

14 

30 

40 

•  7 

+ 

-t- 

-«- 

27 

36 

33 

17 

15 

ao 

27 

42 

40 

+ 

-^ 

-f 

45 

47 

30 

30 

»7 

33 

33 

48 

-H 

+ 

+ 

23 

j$ 

ts 

»5 

18 

— 

30 

a8 

0 

+ 

+ 

298 


Daniel  D.  Jackson 

TABLE  4. — Continued. 


Smith  Solution 


0.1  c.c. 


10  c.c. 


Bile  I,actose 


o.  I  c.c. 


fresh 


Sewage,   outlet   65th    St. 

Brookb-n,  N.  Y.— 

Total  gas 

After  absorption 

Per  cent  carbonic  acid. 

Test  for  B.  Coli 

Water  suspension  No.   i, 

horse  feces — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid. 

Test  for  B.  Coli 

Water  solution  No.  2,  fresh  horse 

feces — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid  .  . 

Test  for  B.  Coli d 

Human  feces  No.  i  kept  in  water 

two  weeks  at  37°  C. — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid.  .  . 

Test  for  B.  Coli 

Human    feces  No.    2    kept 

water  two  weeks  at  37°  C. 

Total  gas 

After  absorption 

Per  cent  carbonic  acid.  .  . 

Test  for  B.  Coli 

Water  suspension   No.   i,    fresh 

human  feces — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid 

Test  for  B.  Coli 

Water  suspension  No.   2,    fresh 

human  feces — 

Total  gas 

After  absorption 

Per  cent  carbonic  acid.  , 

Test  for  B.  Coli 


o 
o 
o 
o 


26 
17 

35 

+ 


15 


42 
28 

+ 


18 


23 


27 
16 

41 


23 


25 
16 
36 

+ 


46 

28 

39 

+ 


44 

25 

43 


26 
19 

27 

+ 


39 
23 

41 

+ 


46 
32 

45 

+ 


40 
28 
30 


41 
25 
39 

+ 


SO 
29 
42 


50 
35 
40 

+ 


27 
20 
26 

+ 


60 
30 
25 

+ 


67 
41 
39 

+ 


37 
37 
35 

+ 


61 
34 

44 

+ 


61 

32 
47 

+ 


32 
22 
31 


In  the  investigation  of  the  relative  efficiency  of  the  Smith  and  lac- 
tose bile  solutions,  275  badly  contaminated  waters  were  examined,  of 
which  65  per  cent  gave  improper  results  with  the  use  of  the  Smith 
solution  while  only  10  per  cent  of  overgrowths  were  found  when  lac- 
tose bile  was  used.  The  total  amount  of  gas  produced  and  the  per- 
centage of  absorption  is  generally  greater  when  lactose  bile  is  used. 
The  gas  usually  forms  somewhat  more  slowly  in  the  lactose  bile,  and 
while  the  second  day's  results  are  preferable  to  those  obtained  by 
the  Smith  solution,  still  better  tests  are  obtained  by  incubating  three 
days. 

CONCLUSIONS. 

The  bile  salts,  and  especially  their  acids,  exert  a  strong  restraining 
action  on  most  species  of  bacteria  which  grow  at  blood  heat. 


Presumptive  Test  for  Bacillus  Coli  299 

This  restraining  actic^n  is  selective.  It  favors  the  increase  of  B. 
coli,  retards  the  growth  of  certain  streptococci,  and  actually  kills  ofT 
the  majority  of  species  which  grow  at  37°  C. 

The  effect  is  due  to  the  cholic  acid  radical  and  is,  therefore,  com- 
mon to  both  of  the  bile  salts. 

Advantage  may  be  taken  of  the  selective  action  of  the  bile  salts  in 
the  determination  of  B.  coli  in  water  by  planting  various  amounts  of 
the  water  to  be  tested  in  bile  lactose  solution.  The  results  are  much 
more  accurate  than  those  obtained  by  the  methods  at  present  em- 
ployed. 

BIBLIOGRAPHY. 

1.  MacConkey.     The  Thompson   Vales  Laboratory  Reports,  3,  Part  I,  p.  41. 

2.  MacConkey.     Ibid.,  Part  II,  p.  151. 

3.  MacConkey.     Ibid.,  4,  Part  II,  p.   151. 

4.  Jordan,  Russell  and  Zeit.     Jour.  Infect.  Dis.,  1904,  i,  p.  682. 

5.  Robin.     Eng.  News,  1905,  54,  p.   160. 

6.  Smith.     Thirteenth  Annual  Report  of  the  State  Board  of  Health  of  New  York, 

1892,  p.   712. 

7.  Smith.     The  Wilder  Quarter-Century  Book,  p.   187. 

8.  Smith.     Centralbl.  f.  Bakt.,  18,  p.  494. 

9.  Prescott  and  Baker.     Jour.  Infect.  Dis.,  1904,  i,  p.  193. 
10.   Irons.     Rep.  and  Papers  Am.  Pub.  Health  Assoc,  26,  p.  311. 


B.  COLI  IN  MARKET  OYSTERS.* 

S.  Henry  Ayers. 
(From  the  Bacteriological  Laboratory,  University  of  Chicago.) 

Since  the  considerable  mass  of  work  on  the  subject  of  typhoid 
fever  and  oyster  infection  has  been  recently  reviewed  by  G.  W.  Fuller,' 
there  is  no  need  of  summarizing  it  in  detail  here.  One  aspect  of  the 
subject,  which  is  of  importance  from  the  public-health  standpoint, 
seems,  however,  to  have  received  little  attention.  Although  oysters 
have  been  taken  from  various  beds  and  examined  for  sewage  pollution, 
I  believe  no  extended  examination  has  been  made  in  this  country  of 
oysters  obtained  from  city  markets.  In  England  Herdman  and 
Boyce"  examined  oysters  from  various  shops,  and  in  a  very  large  pro- 
portion of  cases  B.  coli  was  isolated. 

It  is  a  matter  of  considerable  importance  to  know  how  large  a 
proportion  of  commercial  shell  oysters  on  sale  in  a  given  locality  are 
polluted.  Knowledge  of  this  sort  will  aid  the  health  authorities  of  a 
city  in  detecting  possibilities  of  danger  and  in  drying  up  the  source  of 
infection.  With  this  object  in  view,  examination  has  been  made  of 
shell  oysters  from  a  number  of  the  principal  Chicago  markets. 

It  has  been  well  established  that  the  presence  of  B.  coli  in  oysters 
indicates  sewage  pollution.  C.  A.  Fuller^  has  studied  the  relation 
between  oysters  and  sewage  in  Narragansett  Bay,  and  has  shown  that 
bacterial  analyses  of  oysters  correspond  closely  with  analyses  of  river 
water  above  the  beds  and  with  the  opportunities  for  contamination  as 
determined  by  inspection. 

Considering  B.  coli  as  an  index  of  pollution,  the  examination  was 
carried  on  by  the  following  method. 

Each  oyster  was  opened  with  sterile  instruments,  carefully  removed 
from  its  shell,  and  after  being  rinsed  in  sterile  water  was  placed  in 
a  Petri  dish.  The  oyster  was  then  finely  minced  with  sterile  scissors 
and  mixed  with  5  c.c.  of  sterile  water.  A  dextrose  fermentation  tube 
was  inoculated  with  i  c.c.  of  the  fluid  from  the  minced  oyster.     If  no 

*  Received  for  publication  April  13,  1906. 

'Jour.  FranUin  Inst.,  August,  1903,  p.  81.       '  Thompson-Yates  Laboratories  Rep.,  1899,  2,  p.  43. 
^  Appendix  to  1904  Rep.  V.  S.  Commissioner  of  Fisheries,  pp.  189-238;  Science,  1903,  17,  p.  371. 

300 


B.  CoLi  IN  Market  Oysters  301 

gas  formed  in  the  tube  after  48  hours  at  37°  C.the  test  was  considered 
negative.  However,  if  gas  did  form,  a  litmus  lactose  agar  plate  was 
made  as  soon  as  possible,  and  after  24  hours  agar  tubes  were  inocu- 
lated from  the  red  colonies.  The  pure  cultures  were  then  studied. 
Every  fermentation  tube  not  showing  gas  was  examined  at  the  end  of 
48  hours  for  growth  in  the  closed  arm,  and  a  note  made  of  the  existing 
condition. 

One  c.c.  of  fluid  from  the  oyster  was  thought  to  Ix-  sutlkient  to 
inoculate  the  dextrose  tube  to  show  any  gas-forming  organisms 
present  in  the  oyster.  In  order  to  be  more  certain  of  that  point, 
several  fermentation  tubes  were  inoculated  from  one  oyster,  and  the 
tubes  all  showed  the  same  results.  The  same  process  was  repeated 
on  several  occasions. 

As  far  as  could  be  discovered,  the  majority  of  Chicago  market 
oysters  come  from  Baltimore  and  New  York;  some  come  from  Con- 
necticut.    The  larger  part  of  the  supply  is  from  Baltimore. 

Oysters  from  the  following  markets  were  examined: 

Market  No.  I.     Large  wholesale  and  retail  market.     Supplies  oysters  to  many 
of  the  smaller  markets  in  Chicago.     "Oysters  from  Baltimore." 

Market  No.   II.     Wholesale  and  retail  market.     "Oysters  from   New   York." 
Market  No.  III.     Wholesale  and  retail  market.     "Oysters  from  Ballimon:." 
Market  No.  IV.     Large  retail  market.     "Oysters  from  Connecticut." 
Market  No.  V.     Retail  market.     "Oysters  from  Baltimore." 
Market  No.  VI.     Retail  market.     "Oysters  from  New  York." 
Market   No.    VII.     Retail   market.     "Oysters  obtained  from   wholesale  market 
No.  I." 

Market  No.   VIII.     Retail  market.     "Oysters  from   wholesale  market  No.   I." 
Market  No.  IX.     Retail  market.     "Oysters  from   New  York." 

At  numerous  other  markets  Nnsited  it  was  found  that  the  oysters  were 
obtained  from  some  of  the  wholesale  markets  above  mentioned.  The 
source  of  the  oysters  has  been  recorded  just  as  the  information  was 
received  at  the  markets. 

The  table  following  shows  the  results  of  the  examination. 

As  the  figures  in  the  table  show,  the  oysters  examined  from  market 
No.  I  were  free  from  any  indication  of  sewage  pollution.  Only  four 
dextrose  tubes  from  63  oysters  showed  gas;  from  those  tubes  only 
proteus  forms  were  isolated. 

From  market  No.  II  24  oysters  were  examined.  Two  dextrose 
tubes  showed  gas.     From  one,  an  organism  belonging  to  the  colon 


302 


S.  Henry  Ayers 


Tabulated  Results  of  the  Examination. 


Market 

Number  of 

Oysters 
Examined 

Fermentation 

Tube. 

Growth  in 

Closed  Arm 

after  48  Hours 

But  no  Gas 

Oysters 

Showing 

B.  Coli  or 

Coli-like 

Forms 

Oysters 

Showing 

Proteus 

Forms 

Other  Gas- 
Forming 
Organisms 

No.  I,  March  7 

25 
26 
12 
12 
12 
14 
12 
26 
32 

12 

26 

7 

10 
22 

17 

15 
14 

2 

I 
I 
4 
3 
7 
7 
14 
2 

2 

S 

3 

5 
II 

S 

14 
0 

0 
0 
0 
I 
0 
I 
0 
I 
0 

7 

0 

0 

0 
0 

I 

0 
0 

I 

3 
0 
0 

I 
0 
1 
,    0 
0 

I 

0 

I 

0 

9 

10 

I 

0 

*'          "        14 

23 

No.  II,  "        21 

"         "        26 

No.  Ill,  "       21 

"       "       26 

No.  IV,  "        6 

13 

No.  V,     February  27 

"        March  20 

No.  VI,  February  23 

0 
/  B.  cloacae 

I           2 
0 
f  B.  cloacae 

\           I 

26 

"         March  5 

No.  VII    February  26 

No.  VIII,  March  19 

No.  IX,  March  24 

0 
/  B.  cloacae 

I            I 
0 
0 

group  w^as  isolated,  from  the  other,  a  proteus  form.  From  the  finding 
of  one  coli-like  organism  in  24  oysters  it  could  not  be  said  that  the 
oysters  were  seriously  polluted.  However,  it  seems  to  show  the  possi- 
bihty  of  pollution  and  indicates  that  the  oyster  beds  were  probably 
located  in  a  more  or  less  contaminated  water. 

From  market  No.  Ill  26  oysters  were  examined.  Two  dextrose 
tubes  showed  gas.  One  proteus  and  one  organism  of  the  colon  group 
were  isolated.  The  same  may  be  said  of  that  supply  as  was  said  about 
the  oysters  from  market  No.  II. 

The  same  applies  to  the  oysters  from  market  No.  IV,  of  which  58 
were  examined;   one  coh-hke  organism  being  isolated. 

Regarding  the  oysters  from  market  No.  V,  there  is  no  doubt  that 
the  first  lot,  collected  February  27,  was  badly  sewage-polluted. 
From  seven  of  the  12  oysters  examined  colon  forms  were  isolated. 
The  oysters  did  not  appear  to  have  been  fattened  and  seemed  per- 
fectly fresh.  On  attempting,  some  two  weeks  later,  to  obtain  more 
oysters  at  the  market,  it  was  found  that  the  dealer  no  longer  kept 
shell  oysters  for  sale.  He  explained  that  the  oysters  "went  bad" 
and  opened  before  he  could  sell  them.  The  market  suppHed  oysters,, 
however,  if  they  were  ordered.  On  examination  of  26  oysters  thus 
obtained,  none  of  the  tubes  showed  gas.  It  was  impossible  to  find 
out  just  where  the  oysters  came  from,  except  that  both  lots  came  from 
Baltimore. 


B.  CoLi  IN  Market  Oysters  303 

From  market  No.  VI  39  oysters  were  examined.  No  colon  bacilli 
were  found.     The  supply  was  evidently  free  from  j);)llution. 

The  oysters  from  markets  Nos.  VIll  and  IX  showed  ncj  organisms 
of  the  colon  group. 

The  oysters  from  market  No.  VII,  although  perhaps  not  showing 
indications  of  serious  pollution,  seemd  to  be  in  a  state  of  decomjxjsi- 
tion  which  would  render  them  unsuitable  for  consum[)tion.  From 
17  oysters,  one  organism  of  the  colon  group  and  10  proteus  forms 
were  isolated.  The  oysters  were  no  doubt  old,  as  indicated  by  the 
fact  that  their  shells  were  slightly  open.  The  presence  of  proteus 
forms  probably  shows  that  the  oysters  were  undergoing  decomposi- 
tion, which  view  is  further  borne  out  by  the  putrid  odor  which  accom- 
panied them. 

From  the  results  of  the  examination  it  seems  likely  that  growth  in 
the  closed  arm  of  the  fermentation  tube,  without  gas  formation,  indi- 
cates the  aging  of  the  oysters.  The  presence  of  proteus  forms  prob- 
ably means  old  oysters. 

SUMMARY. 

1.  Eleven  organisms  belonging  to  the  colon  group  were  isolated 
from  the  294  oysters  examined. 

2.  Colon  bacilli  were  found  in  oysters  from  five  markets  out  of  a 
total  of  nine. 

3.  The  presence  of  B.  coli  in  so  large  a  proportion  of  oysters  from 
market  No.  V  seems  to  show  that  the  oysters  had  been  in  contact  with 
sewage.  A  second  lot  of  oysters  from  the  same  market,  but  from 
another  source,  showed  no  evidence  of  pollution. 

4.  Colon  bacilli  in  so  small  a  proportion  of  oysters  in  Chicago 
markets  would  hardly  indicate  a  widespread  jjoUution,  but  a  more 
extended  examination  might  show  different  results. 

5.  The  colon  test  seems  to  afford  a  valuable  means  for  determining 
the  purity  of  a  city  oyster  supply. 

It  is  not  intended  to  present  these  facts  as  a  complete  study  of  the 
oyster  supply  of  Chicago,  but  simply  to  show  the  condition  of  oysters 
from  several  of  the  most  important  markets  in  order  to  illustrate  the 
value  of  such  a  bacterial  examination. 


STUDIES   ON   SIMPLE  AND   DIFFERENTIAL   METHODS 

OF   STAINING   ENCAPSULATED   PNEUMOCOCCI 

IN  SMEAR  AND  SECTION  * 

Augustus    Wadsworth, 

Alonzo  Clark  Scholar  in  Pathology;  Instructor  in  Bacteriology  and  Hygiene,  College  of  Physicians  and 
Surgeons,  Columbia  University,  Assistant  Physician  to  the  Roosevelt  Hospital  Outpatient 

Department. 

{From  the  Department  of  Pathology,  College  of  Physicians  and  Surgeons,  Columbia  University.) 

In  the  course  of  some  experimental  studies  on  pneumococcus 
infection,  the  technic  of  previous  observers  recommended  for  the 
staining  of  the  encapsulated  organisms  was  tested,  and  various 
new  methods  were  devised  in  the  hope  of  securing  rehable  proced- 
ures by  which  pneumococci  may  be  dififerentially  stained  in  cover-glass 
preparations  and  in  sections  of  tissue. 

My  experience  with  these  methods,  old  and  new,  it  is  the  purpose 
of  this  paper  to  record. 

PREVIOUS    METHODS. 

Simple  staining. — In  the  simplef  routine  staining  with  aqueous- 
gentian-violet  or  carbol  fuchsin,  smears  of  pneumococcus  exudates 
may  show  the  organisms  encapsulated;  but  this  is  extremely  uncer- 
tain. Similarly,  under  favorable  conditions  many  of  the  older 
special  methods;|:  devised  for  capsule  staining  often  give  excellent 
preparations,  but  the  results  vary,  and  are  therefore  unreliable 
when  compared  with  those  obtained  with  the  simple,  perfected 
technic  used  by  recent  observers.  The  most  reliable  and  practical 
of  all  these  methods  in  my  experience  are  based  wholly  or  in  part 
on  principles  first  adopted  by  Guarnieri. 

In  1888  Guarnieri 5  made  determinations  of  the  solubility  of 
the  pneumococcus  capsule  in  acid,  alkaline,  and  neutral  salt  solu- 
tions; and  finally  he  obtained  the  proteid  reaction  with  Millon's 
reagent,  thus  indicating  an  albuminous  composition.  On  these 
determinations  Guarnieri  devised  a  new  method  of  staining  the 
encapsulated    organisms    in    exudates:  smears   fixed    in    the    flame 

♦Received    for    publication    February    19,    1906. 

tPane." 

iFriedlander,^   Ribbert.'J   Roux,'*  Muir,"   MacConkey,'  Gordon,*  Kolle  and  Wasserman.' 

304 


Methods  of  Staining  Encapsulated  Pneumococci      305 

were  stained  with  analin-gentian-violet,  then  washed  and  difft-r 
entiated  in  2  per  cent  aqueous  sodium  chloride,  rewashcd  very 
quickly  in  water,  dried,  and  mounted  in  balsam.  Similar  methods 
have  since  been  recommended.  Thus,  Welch'''  adopted  Guar- 
nieri's  method  of  staining,  but  mounted  the  specimen  in  the  sail 
solution,  and  suggested  a  preliminary  treatment  with  glacial  acetic 
acid  on  the  ground  that  the  capsule  was  composed  of  mucin.* 

Buerger*  also  adopted  Guarnieri's  method,  but  recommended 
a  preliminary  fixation  of  the  capsule,  first  in  a  solution  of  chromic 
acid  and  bichloride  of  mercury,  then  in  an  alcoholic  solution  of 
iodine  (U.  S.  P.).  With  Gram's  method  of  staining  this  fixation 
is  essential,  but  in  the  simple  procedures  the  advantage  of  it  is  less 
apparent,  for  practically  the  same  results  are  secured  with  Guar- 
nieri's less  complicated  method. 

Hiss,^  however,  by  substituting  the  ordinary  aqueous-gentian- 
violet  stain  for  the  unstable  anilin-gentian- violet,  and  by  using  0.25 
per  cent  potassium  carbonate  solution,  which  to  some  extent  clears 
the  field,  simplified  and  improved  materially  the  technic  of  capsule 
staining.  Finally,  by  using  a  20  per  cent  copper  sulphate  wash 
instead  of  the  potassium  carbonate  he  found  that  the  specimen 
could  be  dried  and  mounted  in  balsam. 

Formerly  capsules  were  found  only  in  the  exudates  of  infected 
animals,  but  now  they  are  readily  demonstrated  in  organisms  grow- 
ing in  artificial  media.  Boni  found  that  when  cultures  of  pneu- 
mococci are  smeared  in  egg  albumin,  the  capsules  are  easily  stained. 
Hiss  secured  similar  results  with  blood  serum,  and  by  means  of 
his  more  reliable  methods  of  staining  was  able  to  determine  more 
fully  the  importance  of  utilizing  this  principle  in  the  morphological 
study   of  the   pneumococcus. 

By  virtue  of  these  modern  procedures  it  is  now  a  comparalivrly 
simple  matter  to  demonstrate  capsules  on  these  organisms.  It 
is  no  longer  a  question  of  how  encapsulated  pneumococci  may  be 
stained,  but  of  how  they  may  be  most  simply  and  reliably  stained. 

♦Welch  does  not  state  the  reasons  for  this  Ix-lief;  it  Ls  therefore  difRcuh  to  refute  the  p.»iii\-e.  (hou(h 
incomplete,  observations  of  Guamieri.  Mucin  is  a  glycoprotei<l  and  reads  to  Millon'j  rc.\iCent.  hut  ibe 
solubilities  differ,  and  capsules  are  more  constantly  noted  in  albuminous  me<lia  than  in  the  jirrscnce  of 
mucin.  In  fact,  the  mucous  secretions  of  the  mouth  arc  not  a  particularly  favorable  enWronmrnl  (cr  tbc 
demonstration  of  capsules,  whereas  capsules  are  readily  obtained  in  simple  broth,  if  only  the  albumin 
or  even  the  peptones  be  sufficiently  increased. 


3O0  Augustus  Wadsworth 

The  methods  of  Guarnieri,  of  Welch,  and  of  Buerger,  rehable,  though 
in  one  way  or  another  compHcated,  all  give  temporary  mounts, 
and  are  thus  unsatisfactory;  particularly  when  similar  or  better 
results  may  be  secured  by  simpler  procedures  giving  permanent 
preparations  in  balsam,  which  may  be  kept  for  future  reference,  as 
vdth  the  copper  sulphate  method  of  Hiss.^ 

Dijjerential  staining. — These  simple  methods  of  staining  encap- 
sulated pneumococci,  however,  do  not  always  suffice  to  differentiate 
the  pneumococcus  from  other  capsule-forming  bacteria.*  To  com- 
plete this  differentiation  further  steps  are  necessary.  In  pneumonic 
exudates,  pneumococci,  streptococci,  and  pneumobaciUi  (Fried- 
lander)  are  frequently  associated  together.  The  streptococci, 
except  possibly  under  especially  favorable  conditions,  rarely  show 
capsules.  The  pneumobaciUi  not  only  are  encapsulated,  but  the 
short  and  degenerating  forms  are  practically  indistinguishable 
from  the  pneumococci.  The  pneumobacillus,  however,  decolorizes 
by  the  Gram  stain ;  but  this  does  not  show  capsules,  and  when  these 
contaminated  exudates  are  examined  for  the  morphological  deter- 
mination of  the  presence  of  these  species,  both  the  capsule  and  the 
Gram  stains  are  required,  and  even  then  the  observations  may  not 
be  accurately  correlated.  Cultural  characters  are  more  precise  and 
final,  but  require  time;  and,  furthermore,  the  streptococci  and 
pneumobaciUi,  when  present,  usually  predominate;  they  also  grow 
so  rapidly,  and  the  pneumococci  are  frequently  in  such  small  numbers, 
that,  unless  exceptional  precautions  are  taken,  the  pneumococci  may 
not  be  found. 

For  these  reasons  efforts  have  been  made  to  obtain  a  rehable 
method  which  would  demonstrate  the  capsules  and  the  Gram  differ- 
ential in  the  same  preparation.  This  result  has  been  noted  in  speci- 
mens overexposed,  or  exposed  with  heat  to  the  anihn  stain  and  the 
iodine  solutions;  but  this  is  so  exceptional  that  special  methods 
have  been  devised. 

For  this  purpose  Smith's  fixes  the  smears  of  fresh  sputum  from 
pneumonia  patients  in  the  usual  way  by  heat.     The  films  are  then 

♦The  morphological  differences  in  the  capsules  of  the  pneumococci,  as  compared  with  other  encap- 
sulated organisms  resembling  the  pneumococcus,  which  Buerger  obtained  with  his  simple  stain,  and  upon 
which  he  lays  so  much  stress,  depend  chiefly  upon  the  varying  stages  of  development  or  degeneration  and 
solution  of  the  capsule,  and  upon  the  degree  of  decolorization.     They  are  in  no  sense  differential. 


Methods  of  Staining  Encapsulated  Pnelmococci      ;,o7 

steamed  in  anilin-gentian-violet  and  in  the  iodine  solution.  Afkr 
the  usual  alcohol  decolorization  and  a  few  seconds'  exjxjsure  lo 
a  mixture  of  alcohol,  4  parts,  ether,  6  parts,  the  smear  is  counter- 
stained,  first  in  aqueous  eosin,  then  in  Loeflfler's  methylene  blue. 
This  is  followed  by  slight  decolorization  in  95  per  cent  alcohol, 
dehydration  in  absolute  alcohol,  xylol,  and  balsam. 

Buerger*  fixes  the  smears  of  encapsulated  pneumococci  in  M Oi- 
ler's fluid  saturated  with  bichloride,  for  one-half  minute,*  anfl 
washes  in  water.  After  one  minute's  exposure  to  an  alcoh(.lic 
solution  of  iodine  (U.  S.  P.),  and  washing  in  alcohol,  the  smiar 
is  stained  in  the  usual  way  by  the  Gram  method — anilin-gentian- 
violet,  Lugol's  solution,  alcohol  decolorization — and  wa.shed  in 
water.  After  counterstaining  in  aqueous  fuchsin,  the  preparations 
are  washed  and  examined  in  2  per  cent  salt  solution. 

With  these  methods  of  Smith  and  of  Buerger  the  bacterial  cells, 
under  favorable  conditions,  may  be  stained  by  the  Gram  stain  and 
demonstrated  with  capsules.  The  procedures,  however,  are  com- 
plicated, not  as  accurate  and  reliable  as  the  simple  capsule  stains, 
or  give  temporary  mounts;  and  none  of  the  methods  thus  far  con- 
sidered are  applicable  to  the  demonstration  of  encapsulated  organisms 
in  sections  of  diseased   tissues. 

THE   NEW   METHODS. 

In  the  attempt  to  secure  methods  by  which  the  differential  stain- 
ing of  encapsulated  organisms  in  tissues  as  well  as  films  could  be 
easily  accomplished  with  a  reasonable  degree  of  certainty,  a  great 
variety  of  procedures  were  tried  without  success  before  satisfactory 
results  were  finally  obtained.  Suffice  it  to  note  that  all  the  cfi"orts 
to  keep  the  capsules  from  dissolving  in  the  course  of  hardening, 
imbedding,  and  staining,  by  the  addition  of  various  chemicals  to 
the  solutions,  so  successfully  adopted  in  the  simple  methods  of 
Guarnieri  and  of  Hiss  for  films,  failed  to  give  reliable  results.  Excep- 
tionally, a  few  encapsulated  organisms  were  stained  in  sections 
treated  in  this  fashion,  but  the  result  of  these  procedures  proved  to  be 
wholly  beyond  control,  and  was  thus  of  no  practical  value.  It 
was  therefore  evident,  if  the  Gram  stain  was  to  be  used,  that  the 
solution  of  the  problem  depended  primarily  upon   securing   a    per- 

*  In  my  experience  with  this  method  longer  exposures  are  advisable;  in  (act.  eucntial. 


3o8  Augustus  Wadsworth 

manent  fixation  or  coagulation  of  the  capsule.  Obviously,  success 
in  securing  permanent  fixation  depended  upon  the  composition 
of  the  capsule.  In  the  absence  of  data  to  the  contrary,  the  results 
of  Guarnieri's  chemical  studies,  and  the  fact  that  capsule  preser- 
vation or  formation  is  largely  determined  by  the  presence  of  albu- 
min, suggested  with  reasonable  probability  an  albuminous  com- 
position. Attention  was  therefore  directed  to  the  study  of  the 
action  of  bichloride  of  mercury  and  formalin,  which  not  only  pre- 
cipitate albumins,  but  enter  into  chemical  combination  with  them. 

Bichloride  methods. — Bichloride  of  mercury  was  tested  in  both 
aqueous  and  alcoholic  solutions.  Alcoholic  solutions  proved  more 
efficient.  Dried  smears,  fixed  in  saturated  bichloride-alcohol  for 
a  few  minutes  and  washed  in  water,  may  be  stained  in  aqueous- 
gentian-violet,  and  mounted  in  the  usual  way,  or  they  may  be  decol- 
orized by  retreatment  in  the  bichloride-alcohol  for  a  few  seconds 
before  washing  in  water  and  drying.  In  these  preparations  the 
encapsulated  cells  are  often  very  sharply  demonstrated  in  a  per- 
fectly clear  field.  With  the  Gram  differential  procedures  the  cap- 
sules fixed  in  bichloride-alcohol  rarely*  withstand  the  osmosis 
required  to  bring  the  specimen  to  a  balsam  mount,  and  as  appHed 
to  the  study  of  tissues  in  section  it  was  a  complete  failure. 

Bichloride-alcohol  thus  proved  valuable  in  coagulating  and 
partially  fixing  the  capsules,  and  also  in  clearing  the  field;  but, 
aside  from  this,  it  offered  little  practical  advantage  over  the  simple 
staining  procedures  of  other  observers. 

Formalin  methods. — The  fixation  obtained  with  strong  solutions 
of  formalin  proved  more  stable.  It  was  also  found  that,  when 
desirable,  formalin  may  be  effectively  used  as  a  wash  after  simple 
staining  with  aqueous-gentian-violet,  or  5  to  10  per  cent  may  be 
added  to  the  stain  to  shorten  the  procedure.  Although  useful  and 
simple,  these  methods  offer  no  special  advantage  over  the  definite 
fixation  secured  by  the  stronger  (40  per  cent)  solutions  of  formahn,f 

♦Excellent  balsam  preparations  for  demonstration  purposes  were  readily  secured,  but  for  routine 
work  the  results  were  uncertain.  With  other  species  of  encapsulated  bacteria  this  may  prove  more  reUablc 
for  in  sections  of  tissues  hardened  in  bichloride  Wright  and  Mallory"  demonstrated  capsules  on  a  Gram 
negative  organism  resembUng  the  Friedlander  bacillus. 

tThe  addition  of  alkali  to  the  formalin  interfered  seriously  with  the  fixation,  but  the  addition  of 
acetic  add  i  to  2  per  cent  in  some  instances  proved  advantageous,  especially  in  securing  penetration  of  the 
tissues,  and  also  it  was  thought,  by  counteracting  the  alkaline  reaction  of  the  body  tissues. 


Methods  of  Staining  Encapsulated  Pneumococci      309 

after  which  a  great  variety  of  staining  as  procedures,  difTerential 
well  as  simple,  may  be  readily  adopted  according  to  the  character 
of  the  material  to  be  examined,  and  the  purposes  of  the  examination. 
The  simple  routine  gentian-violet  stain  may  be  used,  or  this,  after 
drying,  may  be  decolorized  for  a  few  seconds  in  saturated  bichlo- 
ride-alcohol and  counterstained,  or  the  Gram  difTerential  method 
may  be  employed.  Finally  bits  of  diseased  tissue  may  be  hardened 
in  40  per  cent  formalin,  imbedded  in  celloidin,  and  sections  cut 
and  stained  by  simple  and  differential  methods  to  demonstrate  the 
presence  of  encapsulated  organisms. 

Briefly  tabulated,  the  technic  for  smear  preparations  is  as  follows: 

technic  for  the  fixing  and  staining  of  smears. 


Simple  Stains. 


1  Formalin  40%,  2-5  min.* 

2  Wash  in  water. 

Differential  Stains. 

Gram's  method. 


3  10%  aq.  gentian-violet.  3  Anilin-gentian-violet,  a  min. 

I  I 

4  Wash  water.  4  Blot,  dry.  4  Iodine  solution,  2  min. 

5  Dry,  mount  balsam.    5  Sat.  bichloride-alcohol,  s  sec*  5  Alcohol,  95%  decolori7.e. 


6  Dilute  aq.  eosin  or  fuchsin,  5sec.*    6  Eosin  alcohol.         6  .\q.  (uchsin  (diluted 

7  Blot,  dry,  mount  in  balsam.  7  Oil  of  origanum.    7  Wash  water. 

8  Balsam  mount.      8  Dr)',  mount  in  balsam. 

THE   TECHNIC   FOR   SMEARS   OF   SPUTUM. 

The  demonstration  of  encapsulated  pneumococci  in  smears  of 
exudates  by  these  formalin  methods  is  thus  comparatively  simple. 
The  demonstration  of  encapsulated  pneumococci  in  smears  of 
sputum,  however,  has  proved  in  my  experience  another  problem. 
With  fresh  sputum  containing  exudate  coughed  up  from  the  lung 
good  results  may  often  be  secured,  especially  if  the  more  purulent 
portions  of  the  sputum  are  selected  for  examination.  But  mttrc 
frequently  the  organisms  fail  to  show  good  capsule  development, 
and  in  the  secretions  from  healthy  persons  the  capsule  is  even  less 
marked.  Apparently  saliva  and  mucus  offer  poor  conditions  for 
capsule  formation,  and,  although  the  cells  coming  from  the  lung 
exudates  may  retain  their  capsules  for  some  time,  the  organisms 
growing  in  the  mouth  secretions  may  lose  their  capsules.    To  demon- 

♦Thick  smears  require  longer  exposure  than  thin  smears  of  material  conlaininf  few  cells  and  liiile 

detritus. 


3IO  Augustus  Wadsworth 

strate  capsules  on  such  cells,  albumin*  in  the  form  of  blood  serum 
(Hiss)  or  egg  albumin  (Boni)  must  be  added,  and  thoroughly  mixed 
with  the  sputum  before  the  smears  are  made.  The  mucus  in  which 
many  of  the  organisms  are  imbedded  protects  them  from  the  albu- 
min, and  they  fail  to  show  capsules.  Often  it  is  only  the  isolated 
or  exposed  cells  which  show  capsules.  In  the  fixation  by  formalin 
the  mucus  also  protects  the  cells  from  the  action  of  this  reagent; 
longer  exposure  is  therefore  required,  and  after  this  the  preparation 
should  be  thoroughly  washed  in  water,  or  some  of  the  formalin 
will  be  retained  and  precipitate  the  anilin  stain  when  this  is  used. 

The  demonstration  of  capsules  on  the  pneumococci  in  sputum 
thus  depends  primarily  upon  bringing  the  bacterial  cells  into  an 
albuminous  environment  favorable  to  the  preservation  or  develop- 
ment of  capsules. t  This  accomplished,  the  staining  procedure  is 
purely  a  matter  of  choice.  With  the  formalin  preparations  the 
Gram  stain  may  be  used  and  a  differentiation  for  practical  purposes 
at  once  secured,  as  on  further  study  in  culture,  Gram-positive, 
encapsulated  organisms  of  pneumococcus  morphology  practically 
always  give  the  biological  characters  of  the  pneumococcus.  Occa- 
sionally encapsulated  forms  of  the  Micrococcus  tetragenus  resemble 
atypical  pneumococci,  but  usually  there  is  little  difficulty  in  differ- 
entiating these  forms.  The  Streptococcus  mucosus,  in  my  experi- 
ence, cannot  be  differentiated  morphologically  from  the  pneumo- 
coccus. Streptococci,  in  my  experience,  have  rarely  given  definite 
capsules;};  with  these  formalin  methods. 

THE   TECHNIC    FOR   SECTIONS   OF   TISSUES. 

Although  the  fixation  of  the  pneumococcus  capsule  in  smears 
is  simple,  in  tissues  hardened  for  celloidin  sections  it  is  difficult 
and  less  certain.  The  chief  difficulty  lies  in  securing  sufficient 
penetration.  In  the  body  fluids  the  formaHn  is  diluted,  and  it 
combines  with  the  albuminous  material  which  is  coagulated.   This 

*Blood  serum  seems  to  mix  with  sputum  better  than  egg  albumin,  but  as  suggested  by  Professor 
F.  C.  Wood,  egg  albumin,  if  it  is  diluted  and  made  isotonic  with  NaCl  solution,  will  give  practically  as 
good  results. 

tVVith  some  pneumococci  capsules  are  obtained  with  great  difficulty.  For  complete  discussion  of 
the  dififerentiation  of  the  pneumococci  the  reader  is  referred  to  the  work  of  Hiss,  Borden,   and  Knapps. 

^Occasionally  a  faint,  hazy  periphery,  suggesting  a  shrunken  or  degenerated,  partially  dissolved 
capsule,  was  noted. 


Methods  of  Staining  Encapsulated  Pnei-mococci      311 

protects  many  of  the  bacterial  cells  from  the  action  of  the  formalin, 
and  the  capsules  of  these  cells  are  not  properly  fixed,  and  are  thus 
dissolved  by  subsequent  treatment.  These  difticulties  are  often 
encountered  in  precise  histological  work,  and  are  largely  eliminated 
by  injecting  the  tissue  with  the  hardening  tluid,  or,  when  this  is 
not  feasible,  by  cutting  the  material  into  small  bits  (jr  thin  slices. 
The  lungs  are  easily  injected  through  the  trachea  and  arc  quickly 
hardened  in  the  distended  condition.  The  lesions  may  then  be 
cut  into  small  pieces  and  the  fixation  completed  in  fresh,  strong 
formalin,  the  whole  process  taking  from  three  to  five  hours.  .After 
alcohol  dehydration  the  material  is  imbedded  in  celloidin  and  cut 
in  the  usual  way.  It  is  important  to  have  thin  sections*  for  micro- 
scopical examination;  otherwise  the  encapsulated  bacteria  lying  in 
the  exudate  among  the  cells  cannot  be  easily  distinguished,  .\fter 
alcohol  dehydration  these  sections  may  be  fixed  on  the  slide  by 
partially  dissolving  the  celloidin  with  ether,  or  alcohol  and  ether, 
equal  parts.  A  few  seconds  in  alcohol  will  then  harden  the  thin 
film  of  celloidin  covering  the  section,  and  after  washing  in  water 
the  preparation  is  ready  to  be  stained.  A  variety  of  staining 
procedures  may  be  employed ;t  but  the  Gram  method,  by  virtue 
of  its  differentiation,  has  proved  the  most  useful.  .Anilin-gcntian- 
violet  stain  for  two  to  five  minutes,  iodine  solution  one  to  two  min- 
utes, alcohol  decolorization,  eosin-alcohol  counterstain,  cosin-oil 
of  origanum  to  clear,  and  balsam  mounting,  are  the  several  steps 
of  the  technic.  This  has  rarely  failed  to  give  good  results,  but  occa- 
sionally the  pneumococci,  after  prolonged  exposure,  or  after  expo- 
sure to  weak  or  impure  formalin  solutions,  decolorize  partially  in 
the  alcohol.  This  technical  error,  when  it  occurred,  was  rectititd 
by  using  a  5  per  cent  bichloride-alcohol  for  the  decolorization  fol- 
lowing the  iodine  solution,  so  that  the  material  could  be  studied, 
though  not  so  accurately  as  when  the  cells  were  properly  fi.xed.     By 

*Thin  sections  are  readily  secured  by  paiating  the  surface  of  (he  t)li>rk  with  dilute  cclloidin-rlber 
between  each  stroke  of  the  knife. 

tA  combination  of  the  Nicollc  and  Van  Gieson  methods  of  slainiiiK  hiLS  kImii  some  cxorllcnt  prrra  ' 
rations.  After  staining  in  Locfflcr  alkaline  methylene  blue,  the  dye  Ls  lixcd  in  the  bacterial  rrlLs  by  lo 
per  cent  aqueous  tannic  acid.  This  is  followed  by  a  partial  decolorization  in  alcohol,  cuunterslaininR 
in  van  Gieson 's  strong  fuchsin-picric  acid  solution  (Freelxjrn,  Proc.  N.  Y.  Path.  Soc.  iRoji,  p.  r.t>,  dif- 
ferentiation in  picric  acid  alcohol,  clearing  in  picric  acidnnl  of  origanum,  and  mounting  in  l>aUam.  The 
Gram  stain  may  be  suljstituted  for  the  methylene  blue  and  tannic  acid  slain,  but  it  i<  ap<  to  decoloricc 
in  the  acid  alcohol.     The  picric  acid  cellular  slain  is  more  easily  studied  than  the  eo>in  slain. 


312  Augustus  Wads  worth 

using  strong  bichloride-alcohol  for  decolorization,  Gram-negative 
organisms  retained  the  gentian-violet  stain  and  were  demonstrable 
in  the  sections. 

The  difficulties  of  staining  encapsulated  pneumococci  in  sections 
of  diseased  tissues  are  thus  purely  technical,  similar  to  those  met 
with  in  all  precise  histological  work,  and  with  due  care  easily  elimi- 
nated. By  using  the  Gram  method  of  staining  a  practical  differ- 
ential procedure  is  available  for  the  accurate  determination  of  pneu- 
mococci in  sections  of  diseased  tissues.  This  is  particularly  valuable 
in  the  study  of  pneumonic  lesions,  where  contaminations  or  secon- 
dary infections  often  occur,  and  cultural  examination  fails  to  reveal 
the  true  significance  of  the  bacteria  isolated,  or  the  relationship 
of  these  organisms  to  the  disease  processes. 

These  methods  of  studying  the  encapsulated  pneumococcus, 
uniform  in  principle,  for  the  most  part  simple,  and  adaptable  to 
varying  conditions,  have  been  of  such  value  in  my  studies  that  I 
beheve  they  may  prove  similarly  useful  to  other  workers  in  this  field. 

BIBLIOGRAPHY. 

1.  BoNi.     Miinch.   med.    Wchnschr.,    1900,    47,    p.    1262. 

2.  Buerger.     Med.  News,  1904,  85,  p.  11 17;  Centralbl.  }.  Bakt.,  1905,  orig.   39,  pp. 
216,  335. 

3.  Friedlander.     Fortschr.  der  Med.,   1885,  2,  p.   757. 

4.  Gordon.     Brit.  Med.  Jour.,  March  19,  1904,  p.  659. 

5.  GuARNiERi.     Atti.  d.  V.  Accad.  med.  di  Roma.,  1888,  Ser.  II,  4,  p.  114. 

6.  Hiss.    Jour.  Exp.  Med.,  1904,  6,  p.  335. 

7.  Hiss,  Borden,  and  Knapp.     Ibid.,  1905,  7,  p.  547. 

8.  KOLLE  u.    Wassermann.     Handbuch   der  pathogenen  Mikroorg.,    1903,    Bd.    i, 
p.  422. 

9.  MacConkey.     Lancet,   1898,   2,   p.    1262. 

10.  Mallory.     Zischr.  /.   Hyg.,   1895,   20,  p.   220. 

11.  MuiR  AND  Ritchie.     Manual  of  Bacteriology,   1903,  p.   106. 

12.  Pane.     Centralbl.  f.   Bakt.,    1898,   24,   p.   289. 

13.  RiBBERT.     Deutsche  med.  Wchnschr.,  1885,  11,  p.   136. 

14.  Roux,  NicOLLE  ET  Remlinger.     Traite  de  technique  microbiolog.,  1902,  p.  328. 

15.  Smith.     Boston  Med.  and  Surg.  Jour.,  1902,  147,  pp.  659-67. 

16.  Welch.     Johns  Hopkins  Hospital  Bull.,  1892,   13,  p.   128. 

17.  Wright.     Ztschr.  }.  Hyg.,  1895,  20,  p.  220. 


AN  APPARATUS  FOR  TESTING  THE  VALUE  OF  FUMI 

GATING  AGENTS.* 

Arthur  I.  Kendall, 

Acting  Chief,  Board  of  Health  Laboratory,  I.  C.  C,  Panama. 

CoiNCiDENTLY  wilh  our  incrcascd  knowledge  of  the  part  played 
by  mosquitoes  in  the  spread  of  malaria  and  yellow  fever  there  has 
arisen  a  demand  for  a  class  of  substances  which  shall  be  efiicient  in 
killing  these  insects. 

Although  the  introduction  of  preventive  measures  for  these  dis- 
eases is  a  comparatively  recent  event,  the  number  of  substances — 
"Culicides" — proposed  for  this  purpose  is  very  great;  in  fact,  there 
are  approximately  as  many  culicides  as  there  are  disinfectants  for 
bacteria,  although  our  knowledge  of  the  latter  is  much  the  more 
complete. 

There  is  this  difference,  however,  between  the  two  classes  of  sub- 
stances above  mentioned,  namely  that  whereas  the  bacterial  fumigation 
has  been  studied  with  great  care  and  detail,  with  gradually  perfected 
apparatus  and  methods,  the  mosquito  fumigation  is  still  in  the  rule- 
of-thumb  state,  and  we  have  no  very  definite  data  based  upon  careful 
experimental  procedures  upon  which  to  compare  the  relative 
efficiency  of  different  culicides.  This  is  due,  to  a  considerable 
extent,  to  the  fact  that  at  present  there  is  no  method  or  apparatus 
which  will  allow  such  comparison. 

The  writer  has  had  occasion  to  construct  an  apparatus  for  this 
purpose  which  has  given  satisfaction  in  actual  use,  and  which  has 
furnished  a  ready  means  of  demonstrating  the  applicability  of  the 
various  culicidal  substances  which  from  time  to  time  have  been 
proposed  in  this  connection. 

Before  describing  the  apparatus  in  detail,  it  v.Ill  be  well  to  con- 
sider what  one  must  know  about  a  fumigant;  confining  ourselves  to 
salient  points,  omitting  details  which  are  of  lesser  importance. 

One  must  consider  cost,  availability  (including  continuous  sup- 
ply),   killing    (orculicidal    power),   effect    upon   furnishings   (or   in 

♦Received  for  publication  February  17.  1906. 


314  Arthur  I.  Kendall 

general  upon  material  likely  to  be  exposed  to  its  action),  and  the 
possibility  of  leaving  poisonous  residues.  Of  these  factors,  the  cost 
and  supply  are  factors  quite  without  the  pale  of  any  experimental 
data,  aside  from  the  question  of  cost  in  so  far  as  it  is  affected  by 
questions  of  relative  efficiency,  and  need  not  concern  us  here. 

The  question  of  killing  power,  chemical  changes,  and  action  upon 
substances  exposed  to  the  action  of  the  fumigant  are  points  of  the 
greatest  importance. 

It  should  be  stated  that  there  are  two  factors  involved  in  the 
lethal  action  of  fumigants  upon  mosquitoes:  the  first,  a  stupefying 
effect,  which  is,  or  was,  overlooked  for  a  time,  and  the  actual  death 
of  the  insect.  Almost  invariably  stupefying  precedes  the  death  of 
the  mosquito,  although  the  latter  may  follow  the  former  so  quickly 
as  to  appear  almost  as  a  simultaneous  phenomenon;  hence  it  is 
necessary  so  to  place  insects  upon  which  one  decides  to  try  the  action 
of  a  fumigant  that  they  may  be  freely  exposed  to  the  culicide,  yet 
be  available  for  examination  at  any  period  of  the  experiment. 

The  apparatus  described  below  has  been  constucted  with  these 
points  in  view;  in  addition,  provision  is  made  for  the  introduction 
of  samples  of  the  various  fabrics,  paints,  finishings,  and,  in  general, 
any  sort  of  material  likely  to  be  exposed  to  fumigation. 

The  latter  is  more  important  than  would  at  first  seem  possible. 
For  example,  it  has  been  found  that  certain  paints,  containing  lead 
or  similar  metals,  if  poorly  applied,  or  exposed  to  certain  chemicals 
in  the  presence  of  excessive  moisture,  turn  yellowish,  or  even  dark- 
colored,  due  to  the  formation  of  sulphides.  While  this  does  not 
necessarily  spoil  the  protective  action  of  the  pigmicnt,  it  detracts 
greatly  from  the  esthetic  appearance  and  renders  the  fumigat- 
ing squad  liable  for  damages.  This  is  a  particularly  important 
point  in  tropical  countries,  where  the  humidity  is  always  excessive, 
and  where  much  fumigation  is  necessary. 

The  principle  involved  in  this  apparatus  is  simple.  The  essential 
parts  are  a  box  having  a  content  of  loo  cubic  feet,  provided  with 
a  series  of  holes  through  which  may  be  introduced  cages,  made  of 
wire  gauze  (20  mesh)  six  inches  long,  one  and  one-half  inches  in 
diameter,  closed  at  one  end  with  wire  netting  of  the  same  mesh  as 
that  forming  the  body  of  the  cage,  at  the  other  end  by  a  tapering 


Testing  the  Value  of  Fumigating  Agents  315 

stopper  of  wood,  which  fits  tightly  into  one  of  the  holes  in  the  side 
of  the  box  mentioned  above,  in  such  a  manner  as  to  support  the  cage 
inside  of  the  box  with  the  long  axis  of  the  cage  at  right  angles  lo 
the  wall  through  which  it  projects,  in  which  position  it  is  maintained 
by  the  wooden  stopper.  It  will  be  seen  that  the  stopper  does  three 
things:  it  closes  the  open  end  of  the  netting  cage,  preventing  the 
escape  of  mosquitoes  or  other  insects  which  may  be  inclosed  in  the 
cage,  stops  the  hole  in  the  wall  of  the  box  through  which  the  cage 
is  introduced,  and  keeps  the  cage  in  position. 

Mosquitoes  placed  in  cages,  which  in  turn  are  suspended  from  the 
wall  of  the  box  in  the  manner  above  described,  are  freely  exposed  to 
whatever  may  be  present  in  the  air  within  the  box,  because  the  gases 
or  fumes  pass  freely  through  the  wire  gauze  of  which  the  cage  is 
made;  at  the  same  time,  the  insects  are  imprisoned  in  the  cages, 
and  are  available  for  close  examination  at  any  time  merely  by  remov- 
ing the  stopper,  which  in  turn  removes  the  cage  to  which  it  is  attached. 

The  hole  occupied  by  the  stopper  and  cage  is  closed  by  a  sim- 
ilar stopper,  when  the  cage  is  removed,  thus  preventing  the  escape 
of  gases.  Inasmuch  as  one  may  replace  the  cage  by  a  blank 
stopper  in  a  very  few  seconds,  the  loss  of  fumigating  material  is 
minimal. 

Glass  windows  are  provided  in  opposite  sides  of  ihe  box.  These 
permit  a  limited  examination  of  the  contents  of  the  box  until  the 
fumes  become  too  dense.  The  relative  amount  of  light  coming 
through,  observed  at  the  windows  of  the  box  as  fumigation  progresses, 
furnishes  a  rough  index  of  the  volume,  or  rather  density,  of  the 
fumes  at  any  time.  Hooks  are  provided  in  the  interior  of  the  box, 
upon  which  may  be  hung  various  fabrics;  or  one  may  place  fabrics 
over  the  cages  in  such  a  manner  as  partially  to  shield  the  cages 
containing  mosquitoes  from  the  action  of  the  fumigant. 

In  addition,  a  lamp  (alcohol)  with  a  long  neck  has  been  used 
to  furnish  heat  or  flame  for  any  substances  requiring  the  addition 
of  heat  aside  from  that  produced  by  the  combustion  itst-lf.  The 
lamp  is  supported  by  a  stopper  which  fits  tightly  in  one  of  the  holes 
in  the  bottom,  through  which  the  cages  are  introduced,  and  one 
may  remove  the  lamp  as  one  replaces  the  cages  by  removing  the 
stopper,  which  withdraws  the  lamp,  and  substituting  a  blank  stop- 


3i6 


Arthur  I.  Kendall 


per;  one  may  thus  introduce  or  remove  the  lamp  at  any  time  without 
interfering  with  the  fumigation. 

The  removable  cage  is  especially  important  for  the  study  of  the 
interval  elapsing  between  stupefying  and  actual  death  of  the  mos- 
quitoes. One  places  stupefied  mosquitoes  in  the  fresh  air  for  a 
time,  and  in  this  way  determines  the  interval  necessary  to  produce 
the  various  sets  of  phenomena  between  the  beginning  of  the  experi- 
ment and  the  actual  death  of  the  mosquito. 


FiQ.3. 


Fig.  I. — Front  of  fumigation  apparatus. 

Fig.  2. — Detail  of  door,  showing  overlapping  surface. 

Fio.  3. — Device  for  firmly  closing  door. 

DETAILED   DESCRIPTION  OF  THE   FUMIGATING  APPARATUS. 

The  box  in  which  the  experimental  fumigations  are  carried  on 
is  made  to  contain  exactly  100  cubic  feet,  inside  measure.  The 
dimensions  are  five  feet  wide,  four  feet  long,  and  five  feet  high; 
this  is  approximately  the  average  proportions  of  many  rooms  which 
are  fumigated  in  Panama,  and  these  dimensions  have  been  chosen 
because  it  is  beheved  that  the  ratio  of  height  to  length  and  width 
is  not  without  influence  in  this  work. 

The  material  is  pine,  covered  with  three  coats  of  white  paint, 
to  preserve  the  wood  as  well  as  possible  from  the  action  of  moisture. 
The  door  and  window  in  the  back  are  made  in  such  a  wav  that 


Testing  the  Value  of  Fumigating  Agents 


317 


there  is  three  inches'  overlapping  of  the  door  and  window  ui)on  the 
framing,  to  minimize  the  loss  of  fumigant.  In  actual  practice  we 
have  found  the  leakage  from  this  source  is  practically  w/7. 

The  small  door  in  the  back,  measuring  one  foot  square,  is  so 
arranged  that  one  may  paste  paper  or  any  fabric  over  the  opening 
in  such  a  way  as  to  determine  its  permeability  to  a  given  fumigating 
agent.  It  is  frequently  necessary  to  do  this,  particularly  if  one 
wishes  to  know  the  value  of  such  material  when  emj)l()yefl  for  closing 

5--or H   £• 

■=il 


li. 


Back 


Fig.  4. — Back  view  of  box. 

Fig.  5. — Closing  device  for  door;  the  overlapping  is  the  .same  as  the  front  door. 

openings  in  buildings  which  are  to  be  fumigated.  The  function  of 
the  glass  windows,  each  one  foot  square,  upon  the  sides,  has  already 
been  commented  upon. 

Holes  measuring  one  and  one-half  inches  in  diameter  are  bored 
at  regular  intervals  through  the  bottom,  top,  sides,  front,  and  back 
of  the  box.  Particular  attention  is  paid  to  have  the  alignment 
with  reference  to  the  height  above  the  floor  exact,  because  the  dis- 
tance above  the  floor  is  a  ver}'  important  factor  to  consider  with 
many  fumigating  substances,  particularly  if  the  fumes  are  light, 
and  consequently  very  dense  at  the  top  of  the  room,  ijut  practically 


3i8 


Arthur  I.  Kendall 


K ^-O" 


absent  at  the  bottom.  For  this  reason  holes  are  bored  in  the  floor, 
so  that  mosquitoes  may  be  introduced  and  exposed  at  the  point  of 
minimum  efficiency  of  the  fumigant. 

The  cages  are  composed  of  wire  netting  of  a  mesh  not  less  than 
20  to  the  inch.  This  is  extremely  important;  repeatedly  the  writer 
has  seen  Stegomyia  jasciata  pass  through  a  17-18  mesh  netting. 
One  end  is  closed  with  a  circular  piece  of  the  same  material,  the 
seam  soldered,  and  the  other  end  provided  with  a  band  of  tin,  one 
inch  wide,  which  fits  inside  the^cylinder,  serving  the  twofold  purpose 

of  keeping  the  cylinder  in  shape  at  this 
end  and  furnishing  a  point  of  attach- 
"^  ment  for  the  wooden  stoppers. 
I  The  latter  are  tapered  so  that  they 

1  fit  tightly  inside  the  tin  lining  of  the 
;  wire  cage,  and  at  the  same  time  fit 
tightly  into  the  hole  in  the  side  of  the 
box  through  which  the  wire  cage  just 
passes.  A  half-inch  hole  through  the 
long  axis  of  the  wooden  stopper  per- 
mits the  introduction  of  mosquitoes, 
and  a  cork  stopper  of  appropriate  size 
closes  this  hole  when  the  mosquitoes 
TOP  are  in  place,  preventing  loss  of  mos- 

FiG.  6.-T0P  of  box.  quitoes  or  fumes. 

A  collection  of  wooden  stoppers  which  fit  the  holes  in  the  sides 
of  the  box,  and  which  serve  to  close  these  openings  when  they  are 
^ot  occupied  by  mosquito  cages,  completes  the  outfit. 

The  writer  has  not  only  tested  comparatively  the  ordinary  fumi- 
gating agents,  but  has  made  a  series  of  careful  studies  to  show  the 
relation  of  results  obtained  under  ideal  conditions  as  initiated  in  the 
apparatus  above  described  with  those  obtained  in  large  buildings. 
The  great  amount  of  fumigating  which  is  being  done  in  Panama 
permits  such  comparisons  on  a  practical  scale,  and,  without  going 
too  much  into  detail,  the  results  show  in  general  that  a  slightly  larger 
amount  of  fumigating  agent  per  unit  space  is  required  in  the  relatively 
smaJl  box  than  in  a  building  which  can  be  closed  tightly.  This 
seems  to  be  due,  in  part  at  least,  to  the  proportionately  large  amount 


Testing  the  Value  of  Fumigating  Agents 


319 


of  surface  in  the  box  as  compared  with  that  of  a  very  large  room. 
The  absorption  and  surface  condensation  are  increased  as  the  sur- 
face increases,  and  a  smaller  room,  of  course,  has  relatively  a  larj^er 
surface  than  a  larger  room  of  the  same  general  shajn-. 

Hence,  as  a  rule,  the  fact  that  mosquitoes  are  killed  in  this  appa- 
ratus with  a  certain  amount  of  fumigant  per  unit  space,  under  given 
conditions,  will  hold  for  a  larger  room,  under  the  same  conditions. 

je-. 4'-o" ^ 


'o 


7" 


Sides 


Go5 

Fic.  9. 


Fig.  7. — Section  of  side  showing  one  cage  in  position;  the  Cage  projects  into  the  lumen  of  the  txii 
and  is  closed  on  the  outside  by  a  small  stopper. 

Fig.  8. — Sides  of  box. 

Fig.  g. — One  of  the  wire-gauze  cages  showing  wooden  stopper,  with  hole  for  introductma  oi 
mosquitoes   closed   by  a  wooden   stopper. 

Conversely,  and  even  more  certainly,  if  a  given  concentration  of 
fumigant  will  not  kill  in  the  experimental  box,  in  a  large  space  the 
fumigation,  under  the  same  conditions,  will  be  unsuccessful. 

A  very  interesting  and  important  point  has  come  up  in  con- 
nection with  this  work;  namely,  the  rapidity  with  which  the  aciive 
fumigating  agent  is  evolved  makes  a  great  deal  of  dilTerence  in  the 
efficiency  of  the  fumigation.  For  example,  with  the  less  active 
fumigants  and  culicides,  as  pyrethrum,  the  dilTerence  Ixnwecn  com- 
plete success  and  complete  failure  may  depend  upon  the  ra|)idity 
with  which  the  culicidal  substance  is  evolved  as  smoke.  One  j)ound 
per  1,000  feet  burned  in  three  portions  simultaneously  is  rather  more 


320  Arthur  I.  Kendall 

efficient  than  one  and  one-half  pounds  burned  in  one  lot.     This 
seems  to  hold  true  to  a  lesser  extent  with  all  the  culicides. 

The  amount  of  furniture,  and  in  general  of  objects  which  occupy 
much  space,  diminishes  the  efficiency  of  the  culicidal  action,  and 
should  be  taken  into  account  in  practical  as  well  as  experimental 
fumigations. 


THE     EFFECT     OF    SUBCUTANEOUS    INJECTIONS    OF 

WATER,  RINGER'S  FLUID,  AND  TEN  PER  CENT 

SOLUTIONS  OF  ETHYL  ALCOHOL  UPON 

THE  COURSE  OF  FATIGUE  IN  THE 

EXCISED  MUSCLES  OF  THE 

FROG.* 

Theodore  Hough  and  Clara  Eleanor  Ham 

(From  the  Biological  Laboratories  of  the  Massachusetts  Institute  of  Technology.) 

The  research,  the  results  of  which  are  herewith  reported,  was  sug- 
gested by  a  paper  by  Lee  and  Salant'  on  the  effect  of  alcohol  upon  the 
fatigue  of  skeletal  muscle.  These  investigators,  after  ligaturing  one 
leg  of  a  frog  near  the  hip  joint,  injected  a  lo  per  cent  solution  of  ethyl 
alcohol  into  the  dorsal  lymph  sac  or  the  stomach;  the  ligatured  (nor- 
mal) leg  was  at  once  removed  below  the  ligature,  and  a  fatigue  tracing 
taken  from  the  gastrocnemius,  using  isotonic  contractions  and  giving 
about  60  stimuli  a  minute.  After  allowing  from  20  to  75  minutes 
for  the  absorption  of  the  injected  fluid,  a  similar  tracing  was  taken 
from  the  "alcoholized  gastrocnemius  of  the  other  leg.  Thus  from 
each  animal  records  were  obtained  from  a  non- alcoholized  and  an 
alcoholized  muscle." 

The  results  of  a  large  number  of  experiments  made  by  this  very 
ingenious  method  are  thus  summarized  by  the  authors:  Tracings  from 
the  alcoholized  muscle  showed  "quicker  contraction,  quicker  relaxa- 
tion, larger  number  of  contractions  and  increase  of  work  in  a  given 
time,  larger  number  of  contractions  and  greater  total  amount  of  work 
before  exhaustion  sets  in,  and  delay  of  fatigue;"  and  the  conclusion 
is  drawn  that  "in  medium  quantity  it  (i.e.,  ethyl  alcohol)  e.xerts  a 
favorable  action"  upon  skeletal  muscle. 

We  can  fully  corroborate  Lee  and  Salant's  statement  that  the 
"alcoholized"  muscle  contracts  and  rela.\cs  more  quickly  than  the 

*  Received  for  publication  April  13,   1906. 
«  Lee  and  Salant,  Amer.  Jour,  of  Physiol.,  igoi,  8.  pp.  61-74- 


322  T.  Hough  and  C.  E.  Ham 

muscle  of  the  ligatured  leg.  This  difference  is  seen  in  the  earlier 
contractions  of  the  series  and  becomes  more  pronounced  in  the  later 
contractions.  The  contraction  time  of  the  looth  twitch  of  the  normal 
muscle,  indeed,  may  exceed  one  second,  while  that  of  the  alcoholized 
muscle  may  not  exceed  a  fifth  of  a  second;  and  this  is  specially  true 
of  the  period  of  relaxation,  which  is  often  disproportionally  length- 
ened in  the  "normal"  muscle. 

With  regard  to  the  statement  that  the  alcoholized  muscle  gives  "  a 
larger  number  of  contractions,"  we  have  found  that  a  larger  number 
of  complete  simple  contractions  (i.  e.,  twitches  in  which  relaxation  is 
complete  before  the  next  contraction  begins)  may  be  obtained 
from  the  alcoholized  muscle.  But  it  would  seem  that  Lee  and  Salant 
have  taken  the  disappearance  of  individual  simple  contractions  as  an 
indication  of  the  exhaustion  of  the  muscle.  Figs,  i  and  4  of  their 
paper,  however,  show  clearly  that,  while  the  individual  contractions 
of  the  normal  muscle  do  indeed  cease  sooner  than  those  of  the  alcohol- 
ized muscle,  this  is  due  to  the  fact  that  relaxation  is  not  complete  and 
the  muscle  has  gone  into  tetanus.  Obviously,  such  a  tracing  does  not 
show  exhaustion. 

In  order  to  eliminate  this  disturbing  factor  of  increased  relaxation 
time,  we  have  repeated  the  experiments  with  the  exception  that  in  one 
series  less  rapid  rates  of  stimulation  (one  every  two  or  three  seconds) 
were  used,  and  in  another  series  the  two  muscles  were  thrown  into 
tetanus  and  their  tracings  compared.  In  all  cases  the  alcoholized 
muscles  showed  greater  resistance  to  fatigue;  and  during  the  tetanic 
contractions  the  weight  was  not  only  lifted  to  a  greater  height,  but 
also  held  at  a  greater  height  than  by  the  "normal"  muscle. 

Lee  and  Salant's  results  as  to  the  behavior  of  these  "alcoholized" 
muscles  were,  therefore,  confirmed  in  all  essential  points.  After  the 
absorption  of  the  10  per  cent  alcohol  the  muscle  did  more  work,  and 
the  onset  of  fatigue  was  distinctly  delayed. 

In  all  these  experiments  0.08  c.c.  of  the  10  per  cent  alcohol  per 
gram  of  body-weight  were  injected.  This  means,  for  a  medium- 
sized  frog,  the  addition  of  2 . 5  c.c,  or  more,  of  fluid  to  the  circulating 
medium  of  the  body — by  no  means  an  inconsiderable  quantity  in  this 
animal.     Consequently  it  is  still  an  open  question  whether  the  favor- 


Subcutaneous  Injections  of  Ringer's  Fluid  323 

able  effect  upon  the  working  power  of  the  muscle  is  to  be  allributed 
to  the  ethyl  alcohol,  or  to  the  increase  of  the  circulating  medium. 

In  order  to  test  this,  we  made  parallel,  simultaneous  experiments, 
injecting  into  one  frog  the  solution  of  alcohol,  and  into  another  an 
equivalent  amount  of  water,  or  Ringer's  fluid.  The  operative  pro- 
cedure and  experimental  method  were  essentially  those  of  Lee  and 
Salant:  After  removal  of  the  cerebrum,  with  due  precautions  against 
loss  of  blood,  one  leg  of  each  animal  was  ligatured  and  the  fluid 
injected  into  the  dorsal  lymph  sac.  Simultaneous  tracings  were  then 
taken  from  the  excised  muscles  of  the  ligatured  legs;  and  later  (usually 
about  45  minutes),  simultaneous  tracings  were  taken  from  the  other 
legs.  Edison-Lalande  cells  were  used  to  insure  constancy  in  the 
strength  of  the  stimulating  current,  the  primar)'  circuit  being 
interrupted,  and  the  "  making  "  induction  shock  short  circuited  by  a 
mechanical  "Ablender."  The  rate  of  stimulation  was  usually  al^)ut 
once  every  two  seconds.  In  some  experiments  the  contractions  were 
isotonic,  the  same  weight  being  lifted  by  the  two  muscles  of  the  same 
frog.  In  other  experiments  the  auxotonic  method  was  employed, 
the  muscle  contracting  against  the  resistance  of  an  open,  spiral,  brass 
spring,  accurately  adjusted  so  as  to  insure  equal  initial  tension  and 
the  same  direction  of  pull. 

The  work  done  during  the  same  time  by  two  muscles,  in  fatigue 
tracings  taken  on  drums  revolving  at  the  same,  uniform  speed,  and 
with  the  same  rate  of  stimulation,  is  proportional  to  the  areas  included 
between  the  base  line,*  the  first  and  last  tracings  of  the  given  period, 
and  the  line  joining  the  highest  points  of  the  contraction  records. 
These  areas,  were  therefore  measured  with  a  planimeter,  and  the 
work  done  by  the  two  muscles  readily  compared. 

RESULTS. 

Thirteen  successful  experiments  were  thus  made  upon  the  effects 
of  injecting  the  10  per  cent  alcohol,  and  13  simultaneous,  parallel 
experiments  upon  the  effects  of  injecting  water,  or  Ringer's  fluid. 
The  results  with  each  of  these  fluids  are  given  .separately,  for  con- 
venience, and  the  percentage  of  increa.se  of  work  done  in  the  same 
time  (until  the  appearance  of  marked  fatigue)  is  represented  by  giving 
the  number  of  experiments  in  which  a  given  range  of  increase  occurred. 

*In  our  experiments  the  contractions  relumed  to  the  base  line. 


324  T.  Hough  and  C.  E.  Ham 


TABLE  I. 

The  Effect  of  Injecting  10  per  Cent  Alcohol. 
Percentage  Number  of 

Increase  of  Work  Experiments 

o-io 4 

11-20 2 

21-30 4 

31-40 2 


lOO-IIO I 

Total 13 

TABLE  2. 

The  Effect  of  Injecting  Ringer's  Fluid. 

Percentage  Number  of 

Increase  of  Work  Experiments 

o-io I 

11-20 o 

21-30 I 

31-40 I 

41-50 o 

51-60 I 

61-70 I 

Total 5 

TABLE  3. 

The  Effect  of  Injecting  Water. 

Percentage  Number  of 

Increase  of  Work  Experiments 

O-IO O 

11-20 C 

21-30 2 

31-40 I 

41-50 2 

191-200  I 

Total 6 

It  is  evident  that  the  injection  of  water  and  of  Ringer's  fluid,  as 
well  as  that  of  the  lo  per  cent  alcohol,  was  followed  by  an  increase 
of  work  done.  The  experiments  are  not  sufficiently  numerous  to 
justify  final  conclusions  as  to  the  relative  influence  of  the  alcohol  as 
against  the  water,  or  Ringer's  fluid ;  but  it  is  significant  that  the  per- 
centage of  increase  is  less  after  the  injections  of  alcohol  than  after  the 
injections  of  the  other  fluids.  Thus  while  12  out  of  13  alcohol  experi- 
ments gave  less  than  40  per  cent  of  increase,  5  of  13  with  water  and 
Ringer's  fluid  gave  more  than  40  per  cent.  Moreover,  the  increase 
in  six  of  the  alcohol  experiments  was  less  than  20  per  cent,  while  in 


Subcutaneous  Injections  of  Ringer's  Fluid  325 

only  one  of  the  others  was  it  as  small  as  this.  The  conclusion  is  certainly 
justified  that  the  effect  of  the  injections  of  10  per  cent  alcohol  is  not 
due  to  the  alcohol,  but  to  the  water  in  which  it  is  dissolved;  and  it  is 
even  probable  that  the  increase  of  work  occurred  in  spile  of  the 
presence  of  the  alcohol,  rather  than  because  of  it. 

THE  EFFECT   OF   DOUBLE   INJECTIONS. 

In  order  to  test  still  further  the  conclusions  stated  in  the  last  para- 
graph, we  have  injected,  in  a  certain  number  of  animals,  water  or 
Ringer's  fluid  one  or  more  hours  before  ligaturing  the  leg;  the  sub- 
sequent procedure  was  the  same  as  in  other  experiments — ligature  of 
one  leg,  followed  by  a  fatigue  tracing  therefrom;  then  a  (second) 
injection,  this  time  of  water  or  10  per  cent  alcohol ;  and,  an  hour  later, 
a  fatigue  tracing  from  the  second  leg.  We  desired  to  find  whether 
the  second  injection  of  10  per  cent  alcohol  would  be  without  effect, 
or  show  a  less  marked  effect  after  the  previous  injection  of  water; 
and,  also,  to  find  how  the  effect  of  alcohol  would  compare  with  those 
of  a  second  injection  of  water.     The  results  are  as  follows : 

a)  Water  followed  by  water. — Three  experiments,  which  gave 
increases  of  26.7  per  cent,  6  per  cent,  and  i  per  cent  of  work  by  the 
second  muscle  over  the  work  of  the  first. 

b)  Water  jollowed  by  10  per  cent  alcohol. — Three  experiments,  giving 
increases  of  25 .4  per  cent,  6.9  per  cent,  and  6.5  per  cent. 

c)  Ringer^s  fluid  jollowed  by  alcohol. — One  experiment,  giving  an 
increase  of  1 1 .  i  per  cent. 

d)  Water  jollowed  by  water. — Two  experiments  with  auxotonic 
contractions,  giving  in  one  case  an  increase  of  7.3  per  cent,  and  in 
the  other  a  decrease  of  18  per  cent. 

The  effect  of  the  second  injection  of  water  or  alcohol  is,  therefore, 
much  less  marked  when  it  follows  a  previous  injection  of  water. 
Obviously,  we  should  expect  this  result,  if  the  effect  in  all  cases  is  due 
to  the  addition  of  water  to  the  circulating  medium,  or  to  the  improve- 
ment of  the  circulation  by  increasing  the  volume  of  blood. 

All  the  experiments  of  Lee  and  Salant  were  made  between  Januar>' 
and  June,  and,  therefore,  like  most  laborator)-  experiments  upon  this 
animal,  were  made  upon  winter  frogs,  which  have  been  inactive  for 
several  months,  and  in  which  the  loss  of  water  from  the  bkxxi,  by  the 


326  T.  Hough  and  C.  E.  Ham 

processes  of  excretion,  has  not  been  made  good  by  the  taking  of  food. 
Under  these  circumstances,  the  injection  of  any  fluid  quickly  increases 
the  volume  of  blood,  and  so  improves  the  circulation  through  all 
organs — the  muscles  included.  Perhaps,  also,  by  reducing  the 
osmotic  tension  of  the  plasma,  waste  products  which  have  accumu- 
lated in  the  muscle  during  the  inactive  winter  period  are  removed, 
so  that  the  excised  muscle  becomes  capable  of  greater  work.  This 
obviously  suggests  a  useful  method  of  improving  the  physiological  con- 
dition of  frogs  used  for  laboratory  experiments  during  the  winter 
months. 

SUMMARY. 

1.  Injections  of  water.  Ringer's  fluid,  and  10  per  cent  ethyl  alcohol 
into  the  lymph  sacs  of  a  frog  improves  the  working  capacity  of  the 
skeletal  muscles  and  delays  the  progress  of  fatigue  in  the  isolated 
muscle. 

2.  The  effect  is  largely  independent  of  the  solution  and  seems  to 
be  due  to  the  increase  of  the  circulating  medium,  or  to  the  reduction 
of  osmotic  tension  in  the  blood  plasma,  or  to  both  of  these  causes 
combined. 

3.  The  improvement  in  the  working  power  of  the  muscle,  which 
Lee  and  Salant  observed  after  injections  of  10  per  cent  alcohol,  is 
not  due  to  the  alcohol. 


NOTES  ON  A  CASE  OF  APPARENT  PULMONARY  TLHKK 
CULOSIS  ASSOCIATED  WITH  THE  CONSTANT 
PRESENCE  OF   DIPHTHERIA-LIKE 
ORGANISMS  IN  THE  SPUTUM  * 

Burt  Ransom  Richards, 
Director,    Boston    Board    of    Health    BacterioloRical    Laboratory. 

On  January  8,   1906,   a  sample  of  sputum   from   H S 

was  submitted  to  this  laboratory  in  one  of  the  regular  outfits  to  be 
examined  for  the  bacilli  of  tuberculosis.  In  the  course  of  the  rigular 
routine  the  specimen  was  stained  in  steaming  carbol-fuchsin  for 
five  minutes,  decolorized  with  acid-alcohol  (5  per  cent  HCl),  and 
counterstained  with  Loeflfler's  methylene  blue.  When  examined 
under  the  microscope,  no  bacilli  of  tuberculosis  were  found,  but 
the  specimen  contained  large  numbers  of  bacilli  which  mor- 
phologically were  indistinguishable  from  the  bacillus  of  diphtheria. 
In  fact,  no  other  species  of  organisms  were  apparent  in  thi'  speci- 
men, microscopically.  As  is  usual  in  such  cases,  the  attending 
physician  was  requested  to  secure  a  second,  and  in  this  case  uncar- 
bolized,  specimen.  The  second  specimen  was  received  on  January 
15,  1906,  and  from  it  the  organisms  in  question  were  isolated,  but 
not  without  some  diflSculty,  owing  to  the  presence  of  a  large,  rapidly 
growing  coccus  which  tended  to  spread  and  overgrow  the  di|)h- 
theria-like  organism.  The  cultural  characteristics  of  the  organism 
were  then  studied  as  follows: 

Morphology. — Indistinguishable  from  the  Klebs-Loeffler  organ- 
ism. A,  C,  and  D  types  (Wesbrook)  present;  C  types  predt)mi- 
nating.  No  spores.  Stains  by  the  ordinary  stains,  and  by  Gram's 
and  Neisser's  stain. 

Hanging-block. — The  post-fission  movement  called  "snapping,"  and  whiih 
appears  to  be  characteristic  of  the  diphtheroid  group,  was  here  obser\-ed.' 

Agar    stroke. — Filiform    growth    at    first,    later   slightly    plumose;  elevation    fla 

with   slightly   raised   edges.     Luster  glistening.     Topography   smooth   at   first,    later 

slightly  contoured.     Opaque,  non-chromogenic.     Consistency,  butyric.     Medium  not 

discolored. 

♦Received  for   publication   April   7,    1006. 

•Hill  and  Rickards.    Rep.  and  Papers,   Amer.  Pub.  Health  .\ssch  ,  j8,  p.  470- 

327 


328  Burt  Ransom  Rickards 

Potato. — Grows   well,   but   is   invisible. 

Blood  serum. — Growth  as  described  under  agar  stroke,  except  that  it  grows 
more  luxuriantly,  spreads  slightly,  and  is  faintly  flesh-colored.     Typical  appearance. 

Agar  stab. — Filiform,  spreading  slightly  on  surface. 

Gelatin  stab. — Filiform;  no  liquefaction;  slight  growth  on  surface. 

Broth. — Surface  growth,  none;  tubidity,  slight;  sediment,  considerable,  granular 
— adhering  to  glass. 

Milk. — Coagulation,  none;    consistency,  unchanged. 

Agar  colonies. — Punctiform;  edge  entire;  finely  granular. 

Relative  growth  at  20°  and  jy°. — Grows  well  at  37°  C;  very  slow  growth  at  20°  C. 

Fermentation  of  dextrose.* — No  gas  production;  no  growth  in  closed  arm;  acidi- 
fying coefficient:     i  day,  — ;    2  days,  ^^;   4  days,  ^-^;    6  days,  — ;  8  days,   ^-^; 

10  days,  — . 

Fermentation  of  lactose.* — No  acid  or  gas  production. 

Fermentation  0}  saccharose.* — No  acid  or  gas  production. 

Indol  production. — Very  slight. 

Pathogenesis. — Very  slight.  A  guinea-pig  inoculated  wath  i  per  cent  of  his 
body-weight  of  a  0.2  per  cent  dextrose  broth  grown  10  days  at  37°  C.  showed  a 
very  slight  edema  at  site  of  inoculation  and  slight  injection  of  suprarenals  in  two  days. 

For  the  following  clinical  history  the  writer  is  indebted  to  Dr. 
A.  H.  Bassett  the  attending  physician : 

Patient,  Miss  H —  S — .  Age,  32  years;  medium  height;  sandy  complexion; 
slightly  stoop-shouldered;  weight,  112  pounds.  Occupation,  clerk.  First  consulted 
physician  on  December  30,  1905.  Symptoms:  loss  of  flesh,  pain  in  upper  chest 
and  right  shoulder,  loss  of  appetite. 

Present  illness  dates  back  five  years,  previous  to  which  patient  said  she  had  no 
sickness  since  childhood.  No  history  of  diphtheria.  First  symptoms  were  decided 
hoarseness,  aggravated  by  dampness  or  by  becoming  over-tired.  For  past  two  years 
the  amount  of  perspiration  has  been  over  normal.  Regular  night  sweats  eight  months 
ago;  not  as  profuse  at  present  time.  Took  care  of  consumptive  sister  one  year  ago. 
Symptoms  gradually  growing  worse  since  then.  Some  expectoration,  but  not  abun- 
dant. Within  the  past  year  urine  has  at  times  contained  some  blood.  This  condi- 
tion lasts  for  some  time  and  then  passes  off.  The  patient's  urine  was  examined  by 
one   physician   who   reported   "nothing   pathologic."     No  urine   analysis  by   writer. 

Physical  examination  (reported  by  Dr.  H.  C.  Clapp):  "Small  area  of  dulness 
in  apex  of  right  lung,  heard  more  posteriorily;  broncho-vesicular  respiration;  bron- 
chophony; normal   temperature.     CHnically,    tuberculosis." 

On  February  26,  six  weeks  after  the  last  previous  sputum  was 
examined,  a  third  specimen  was  requested  and  obtained,  in  order 
to  demonstrate  the  continued  presence  of  the  diphtheria-like  organ- 
ism. No  tubercle  baciUi  were  found,  but  the  above  organism 
was  present  in  undiminished  numbers. 

♦0.2  per  cent,  in  sugar  free-broth. 


I'LAIK   3. 


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Case  of  Apparent  Pulmonary  Tuberculosis  329 

Three  separate  examinations  have  thus  been  made  on  three  samples 
of  sputum  submitted  at  intervals,  and  in  each  case  no  tubercle  bacilli 
were  found  after  an  exhaustive  search.  This  fact  in  itself  does 
not,  of  course,  rule  out  the  possibility,  but  lessens  by  so  much  the 
probability,  of  their  presence  in  the  lung  tissue.  On  each  of  these 
examinations  an  organism  resembling  B.  diphtheriae  both  morpho 
logically  and  culturally  has  been  present  in  large  numbers. 

The  fact  of  the  continued  presence  of  this  organism  raises  a 
suspicion  in  the  mind  of  the  writer  as  to  whether  the  organism, 
if  not  the  primary,  may  not  at  least  be  a  contributing  factor  in 
producing  the  symptoms  above  described. 

Dr.  Louis  Hoag,  of  the  Danvers,  Mass.,  State  Hos|)ital,  has 
in  a  number  of  cases  isolated  diphtheria-like  organisms  from  the 
lungs  of  patients  who  had  died  of  broncho-pneumonia.  Dr.  Hoag 
agrees  with  the  writer  that  these  organisms  differ  in  many  respects 
from  the  one  here  described. 

The  writer  is  unaware  of  any  previous  descriptions  in  the  liter- 
ature analogous  to  the  above.  Dr.  W.  H.  Smith,  of  this  city,  has, 
however,  very  kindly  submitted  notes  on  a  case  which  came  under 
his  care  at  the  Massachusetts  General  H()s[)iial  in  1904.  The 
clinical  history  bears  a  most  decided  resemblance,  including  a  family 
history  of  tuberculosis,  tubercular  symptoms,  cough — four  years — 
moist  rales,  apex  of  right  lung,  etc.  Sputum  examinations  show  no 
bacilli  of  tuberculosis,  but  the  constant  presence  of  an  organism 
resembling  the  diphtheria  bacillus  morphologically,  but  of  very 
low  virulence. 

It  is  the  intention  of  the  writer  to  fi)llow  the  two  cases  cited,  if 
circumstances  permit,  with  a  view  to  determining,  if  po.ssible,  the 
role  played  by  the  diphtheria-like  organisms. 

The  writer  wishes  to  acknowledge  his  indebtedness  to  Dr.  V.  H. 
Slack,  Assistant  Bacteriologist,  for  help  in  isolating  and  identifying 
the  organism. 

DESCRIPTION  OF  PLATK  3. 

Pure  culture,  diptheria-likc,  organism  isolated  from  si)Utuni.  Twrnty-four-hour 
growth  in  serum.      Methylene  blue.  X   1500. 


If.  C.  State  Coli«^< 


